BTNL3 / 8-targeted constructs for payload delivery to the gastrointestinal system

Abstract

Protein constructs comprising a BTNL3 / 8 targeting moiety, a payload, and an optional linker are described. Pharmaceutical compositions comprising the constructs and methods of use are also presented. [Selection diagram] None

Description

[Technical field]

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

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

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

[0004] Tissue-selective homing of T cells is considered a key element during the integration of normal immune responses. One interesting and highly unique class of such “tissue-homing” or “tissue-resident” T cells is γδ T cells. γδ T cells are highly compartmentalized T cells, showing subset localization to specific tissues, e.g., murine Vγ5 T cells are found exclusively in the epidermis, whereas murine Vγ7 T cells are specific to the intestine. Some murine and human IEL compartments depend for their development and survival, through mechanisms that remain unclear, on proteins expressed by epithelial cells at rest, such as the butyrophilin (BTN) and butyrophilin-like (Btnl / BTNL) genes. (Di Marco Barros et al., Cell. 2016; 167(1): 203-218; Kabelitz et al., F1000 Faculty Rev: 782; June 5, 2017).

[0005] Butyrophilins and butyrophilin-like proteins (BTN / BTNL) are a family of immunoglobulin superfamily members that influence immune, such as T cell selection, and developmental processes, such as differentiation and cell fate determination (Arnett and Viney, Nature Reviews, 2014(14)pp. 559-569). BTNL proteins (specifically BTNL3 and BTNL8) are disproportionately highly expressed in enterocytes of the human intestine, and it has been shown that BTNL3 / 8 can specifically regulate human Vγ4+γδT cells (Di Marco Barros et al., Cell. 2016; 167(1): 203-218). The BTN and BTNL proteins have been reported to regulate multiple T cell responses in either a negative or positive manner, but it is unclear how such signals are transmitted to T cells (Kabelitz et al., F1000 Faculty Rev: 782; June 5, 2017).

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

[0007] (4. Overview) To further explore this (albeit poorly characterized) interesting tissue / immune compartment "shaping" and "homing", we sought to explore the mechanism by which γδ T cell tissue targeting and specification is achieved. First, we attempted to engineer a panel of recombinant heterodimeric and homodimeric γδ T cell receptor proteins (TCRs) starting from the human γ and δ chain sequences. To achieve this, we explored a variety of fusion partners and confirmed that the resulting recombinant heterodimerized and homodimerized γδ chains could be expressed (e.g., in HEK293 cells), remained intact, and underwent little aggregation upon purification (as measured by size exclusion chromatography). 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 TCR α-β chain constant domain partners in combination with engineered leucine zippers (Xu et al., PNAS, 2011 Vol. 108; pp. 2414-2419). However, while these successful experiments were not exhaustive, one skilled in the art would now recognize that they would be motivated to identify alternative approaches to generate recombinant γδ homodimers, heterodimers, and monomeric subunits.

[0008] We created a panel or library of T cell receptor recombinant proteins derived from this human γδ sequence and then used it to explore which (if any) recombinant γδ pairs exhibit any tissue specificity or selectivity. Therefore, we incubated these proteins with cell lines overexpressing BTNL3 / 8. Unprecedentedly, through controlled comparative studies, we identified certain recombinant T cell receptors (e.g., recombinant γ4δ1TCR and recombinant γ4δ2TCR) that specifically recognized BTNL3 / 8 proteins, while others (e.g., recombinant γ2δ1TCR and recombinant γ8δ1TCR) did not. This shows, for the first time, that γ4 containing γδTCRs directly interact with BTNL3 / 8 expressed on the cell surface.

[0009] Furthermore, TCR deep sequencing showed that there was selective enrichment of γδ T cells expressing γ4 TCR within the BTNL3 / 8-responsive fraction: the sequencing data and recombinant TCR experiments demonstrated that TCR pairings involving the γ4 subunit selectively bind to BTNL3 / 8.

[0010] Thus, in a first aspect, a protein construct is provided, the protein construct comprising a BTNL3 / 8 targeting moiety, a payload, and an optional linker connecting the targeting moiety to the payload.

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

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

[0013] In one embodiment, the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. In one embodiment, the Vγ domain CDR3 comprises a human Vγ4 CDR3 sequence. In one embodiment, the Vγ domain CDR3 comprises a human Vγ2 CDR3 sequence. In one embodiment, the Vγ domain CDR3 comprises a mouse Vγ7 CDR3 sequence. In one embodiment, the J region is a Vγ J region. In one embodiment, the J region is a mouse Vγ J region. In an embodiment, the J region comprises a SEQ ID NO: selected from the group consisting of SEQ ID NOs: 15-18.

[0014] In an embodiment, the BTNL3 / 8 targeting component of the protein construct further comprises a paired Vδ domain. In one embodiment, the Vγ domain and the Vδ domain are covalently linked by at least one disulfide bond. In one embodiment, the Vγ domain and the Vδ domain are paired by a specific heterodimeric interaction. In one embodiment, the heterodimeric interaction is leucine zipper complementarity. In one embodiment, the targeting component comprises SEQ ID NO:9. In one embodiment, the targeting component is SEQ ID NO:10. In one embodiment, the targeting component is SEQ ID NO:11. In one embodiment, the targeting component comprises a single-chain in-frame fusion of a Vγ domain and a Vδ domain. In one embodiment, the Vγ domain is N-terminal to the Vδ domain. In one embodiment, the Vγ domain is C-terminal to the Vδ domain. In one embodiment, the single-chain in-frame fusion of a Vγ domain and a Vδ domain comprises an internal linker sequence. In one embodiment, the Vδ domain is a human Vδ domain. In one embodiment, the human V5 domain is V51, V52 or V55. In one embodiment, the human V5 domain is V51.

[0015] In one embodiment, the protein construct further comprises a first T cell receptor constant region, which is fused in-frame to the C-terminus of the Vγ domain. In one embodiment, the first T cell receptor constant region is a human T cell receptor constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor β constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor α constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor γ constant region. In one embodiment, the targeting moiety further comprises a second T cell receptor constant region, which is fused in-frame to the C-terminus of the paired Vδ domain. In one embodiment, the second T cell receptor constant region is a human T cell receptor α constant region. In one embodiment, the second T cell receptor constant region is a human T cell receptor β constant region. In one embodiment, the second T cell receptor constant region is a human T cell receptor δ constant region. In one embodiment, the in-frame fusion of the Vδ domain and the second T cell receptor constant region comprises an internal linker sequence between the Vδ domain and the second T cell receptor constant region.

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

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

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

[0019] In an aspect, described herein is a method of treating a gastrointestinal condition in which gastrointestinal tissue expresses BTNL3 / 8, comprising administering a therapeutically effective amount of a pharmaceutical composition according to any one of claims 62-66 to a patient suffering from a condition in which gastrointestinal tissue expresses BTNL3 / 8. In one embodiment of this method, the payload of the protein construct is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an anti-inflammatory cytokine. In one embodiment, the anti-inflammatory agent is an anti-pro-inflammatory agent. In one embodiment, the anti-inflammatory agent is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is prednisone. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload is an immunomodulatory agent.

[0020] In an aspect, described herein is a method of treating inflammatory bowel disease comprising administering to a patient suffering from inflammatory bowel disease a therapeutically effective amount of any of the pharmaceutical compositions described above. In one embodiment, the inflammatory bowel disease is ulcerative colitis. In one embodiment, the inflammatory bowel disease is Crohn's disease. In one embodiment, the payload of the protein construct is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an anti-inflammatory cytokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or transforming growth factor beta (TGFβ). In one embodiment, the anti-inflammatory agent payload is an anti-pro-inflammatory agent. In one embodiment, the anti-inflammatory agent payload is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is prednisone. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload of the protein construct is an antibiotic. In one embodiment, the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin or amoxicillin. In one embodiment, the payload of the protein construct is a calcineurin inhibitor. In one embodiment, the calcineurin inhibitor is cyclosporin A or tacrolimus. In one embodiment, the payload of the protein construct is an immunomodulator. In one embodiment, the immunomodulator is an immunosuppressant. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate or a thiopurine. In one embodiment, the payload of the protein construct is a protein payload. In one embodiment, the protein payload is an antibody, an antibody fragment or a single chain variable fragment. In one embodiment, the protein payload comprises and at least one ABS specific for the TNFα antigen. In one embodiment, the protein payload comprises the complementarity determining regions (CDRs) of adalimumab, infliximab, or certolizumab.In one embodiment, the protein payload comprises at least one ABS specific for an interleukin antigen. In one embodiment, the interleukin is IL-12, IL-23, or a combination thereof. In one embodiment, the protein payload comprises the CDRs of ustekinumab or brikinumab. In one embodiment, the biologic payload comprises at least one ABS specific for an integrin antigen. In one embodiment, the integrin is alpha4 integrin. In one embodiment, the protein payload comprises the CDRs of infliximab, natalizumab, or vedolizumab. In one embodiment, the protein construct comprises an analgesic payload. In one embodiment, the protein construct comprises a nutraceutical payload.

[0021] In some aspects, described herein is a method for treating irritable bowel syndrome comprising administering to a patient suffering from irritable bowel syndrome a therapeutically effective amount of any of the above pharmaceutical compositions. In some embodiments, described herein is a method for treating diverticulitis comprising administering to a patient suffering from diverticulitis a therapeutically effective amount of any of the above pharmaceutical compositions. In some embodiments, the payload is an antibiotic. In some embodiments, the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin.

[0022] In some aspects, described herein is a method of treating celiac disease comprising administering to a patient suffering from celiac disease a therapeutically effective amount of any of the pharmaceutical compositions described above. In some embodiments, the payload is an immunosuppressant. In some embodiments, the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine.

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

[0024] In some aspects, described herein is a method for treating a metabolic disorder or deficiency comprising administering to a patient suffering from the metabolic disorder or deficiency a therapeutically effective amount of any of the pharmaceutical compositions described above. In some embodiments, the payload is a nutritional supplement. In some embodiments, the nutritional supplement is an enzyme or a vitamin.

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

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

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

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

[0029] FIG. 3B shows expression 72 hours after transduction of TCR constructs with cloned CDR3 pairs derived from γδ TCR variable domains in TCR-deficient Jurkat (J76 cells).

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

[0031] Figure 4B shows that three independent Vy4V51 transduced J76 lines responded differently to BTNL3 / 8 expressing cells than Vy9V52 lines. B3, C11 and H7 represent three different CDR3 pairs obtained by the method shown in Figure 2.

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

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

[0034] Figure 6B shows previously published structures of Vy4 / V51 and Vy5 / V51 paired variable domains aligned using Cn3D. The CDR4 regions form appropriate loops that are indistinguishable from conventional CDRs. Of note, the Vy4 and Vy5 CDR4 loops show significant conformational differences.

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

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

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

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

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

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

[0041] FIG. 11B is a schematic diagram showing BTNL3+BTNL8 expressing cells incubated with soluble His-tagged TCR and anti-His tag antibody at 37° C.

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

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

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

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

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

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

[0048] FIG. 16B shows imaging cytometry results of DMEM or trypsinized 293T.L3L8 cells incubated with soluble TCR+α-His antibody complex at 4° C.

[0049] FIG. 16C shows imaging cytometry results of DMEM or trypsinized 293T.L3L8 cells incubated at 37° C. with soluble TCR+α-His antibody complex.

[0050] The above drawings depict various embodiments of the present invention for purposes of illustration only. Those skilled in the art will readily recognize from the discussion below that alternative embodiments of the structures and methods shown herein may be employed without departing from the principles of the present invention as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] 6. Detailed Description (6.1.Definition) Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings set forth below.

[0052] As used herein, a "protein construct" refers to one or more polypeptide chains that contain at least two functional elements, a BTNL3 / 8 targeting moiety and a payload, which may or may not be a protein payload.

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

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

[0055] As used herein, "Vγ domain" refers to the variable domain of the T cell receptor (TCR) gamma chain. This Vγ domain includes the J region and the complementarity determining regions (CDRs), CDR1, CDR2, CDR3 and CDR4. The numbering of Vγ domain residues is shown in FIG. 1, where residue 19 is the N-terminal amino acid of the mature Vγ domain after signal sequence cleavage. For Vγ domains not shown in FIG. 1, residue numbering is assigned after best alignment to the sequence in FIG. 1. A "corresponding residue" of a Vγ domain is the amino acid at the same numbered position as the designated corresponding residue.

[0056] As used herein, "Vγ domain CDR4" refers to a continuous 16 amino acid portion of a Vγ domain located between the CDR2 and CDR3 regions, and corresponds to amino acid sequence positions 85-100 in FIG. 1. The amino acid sequence of CDR4 of a human Vγ4 domain has the sequence of SEQ ID NO:3. The amino acid sequence of CDR4 of a human δγ2 domain is SEQ ID NO:5. The amino acid sequence of CDR4 of a mouse Vγ7 domain is SEQ ID NO:4.

[0001] As used herein, "payload" refers to any molecule that is delivered to a target cell of interest (e.g., a cell expressing BTNL3 / 8 and / or an intestinal epithelial cell). The payload can include nucleotides, nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or that inhibit transcription), nucleic acids such as DNA and RNA (e.g., mRNA, RNAi, miRNA, siRNA, snRNA, snoRNA, piRNA, exRNA, scaRNA, and lncRNA), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or that inhibit translation), polypeptides (e.g., enzymes, biologics), lipids, carbohydrates, small molecules (e.g., small molecule drugs and small molecule toxins), and combinations thereof. In some embodiments, the payload is a therapeutic agent. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In some embodiments, the payload is an antibody.

[0057] As used herein, "linker" refers to any molecule that can be used to link functional elements of a protein construct (e.g., targeting moieties and payloads). In some embodiments, linkers can be used to allow site-specific conjugation of molecules to functional elements of a protein construct (e.g., targeting moieties and payloads). In some embodiments, linkers can be used to identify or detect protein constructs in vitro or in vivo.

[0058] As used herein, a "peptide linker" refers to a linker that is a polypeptide. In some embodiments, the peptide linker is fused in frame to a functional element of a protein construct (e.g., a targeting moiety and a payload). In some embodiments, the peptide linker allows for site-specific conjugation of a molecule to an element of a protein construct. The peptide linker can be any length and type of amino acid sequence that allows for the desired conformation of the functional element to which it is fused.

[0059] As used herein, an "internal linker" refers to a polypeptide sequence that is covalently linked at both its N-terminus and C-terminus to at least one additional polypeptide. In one embodiment, the internal linker is a polypeptide chain within the targeting moiety (e.g., an internal linker between a Vγ domain and a Vδ domain). The internal linker may be any length and type of amino acid sequence that allows the additional polypeptide to which it is fused to maintain the required conformation.

[0060] As used herein, "antibody" includes any antibody protein construct comprising at least one antibody variable domain that comprises at least one antigen binding site (ABS). Antibodies include, but are not limited to, variable domain-only molecules, single chain variable fragments (scFv), single chain Fab fragments (scFab), bispecific antibodies, hybrid IgG, Fab fusion proteins, Fc-modified IgG, adducted IgG, diabodies, single chain diabodies, DARTs, tandem diabodies (tandAbs) and minibodies.

[0061] As used herein, "single-chain in-frame fusion" refers to a single-chain in-frame fusion T cell receptor variable domain (scTv), in which at least a portion of at least two polypeptide variable domains of a T cell receptor are produced as a single fusion polypeptide chain, the variable domain sequences of which are fused in-frame. A single-chain in-frame fusion T cell receptor variable domain (scTv) can include more than one variable domain and / or constant region and may be paired with a T cell receptor of any origin. Thus, scTv includes tandem scTvs in which two or more variable domains are fused in-frame.

[0062] As used herein, "antigen-binding site" (ABS) refers to the region of an antibody molecule that specifically recognizes and binds to a given antigen or epitope. An ABS is said to bind to a specific antigen or epitope with a particular affinity. "Affinity," as described herein, refers to the strength of interaction of non-covalent intermolecular forces between one molecule and another. Affinity, i.e., the strength of the interaction, is determined by the dissociation equilibrium constant (K D ) and low K D The K value indicates that the interactions between the molecules are strong. D The affinity is measured by methods well known in the art, including but not limited to biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays. The affinity between an ABS and its cognate antigen or epitope is measured by a factor of 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than M D It has a value.

[0063] As used herein, "small molecule" refers to a molecule having a low molecular weight of less than 900 Daltons and does not include peptides. Small molecules may 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 that has anti-inflammatory activity, including, but not limited to, interleukins (IL) IL-1ra, IL-4, IL-6, IL-10, IL-11, IL-13, and transforming growth factor beta (TGFβ).

[0065] As used herein, "anti-inflammatory agent" refers to any molecule or agent that inhibits the activity or expression of a proinflammatory cytokine. Anti-inflammatory agents include inhibitors of proinflammatory cytokines, including, but not limited to, interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor alpha (TNFα), interferon gamma (INF-γ), and granulocyte-macrophage colony stimulating factor. Anti-inflammatory agents include soluble cytokine receptors with anti-inflammatory activity, such as soluble tumor necrosis factor receptor p55, soluble tumor necrosis factor, p75, soluble IL-1 receptor type II, IL-18 binding protein, etc. Anti-inflammatory agents also include cytokine receptors that lack intracellular signaling that competes with proinflammatory cytokine receptors, such as membrane-bound IL-1 receptor type II.

[0066] As used herein, "immunomodulatory agent" refers to any molecule that alters the immune response and / or activity of cells of the immune system. In certain embodiments, an immunomodulatory agent is an immunosuppressant or an immunostimulatory agent. "Immunosuppressant" refers to any molecule or agent that decreases the immune response and / or activity of cells of the immune system, while "immunosuppressant" refers to any molecule or agent that increases the immune response and / or activity of cells of the immune system.

[0067] As used herein, "immune-related condition" refers to any condition or disorder associated with an alteration in the activity of the immune system. Immune-related conditions include conditions associated with an increased or decreased immune response. Immune-related conditions include, but are not limited to, autoimmune diseases, inflammatory conditions, allergic reactions, immune deficiencies, hematopoietic cancers and other hematopoietic disorders.

[0068] As used herein, "inflammatory condition" refers to any condition or disorder associated with the presence or increase of inflammation in inflamed tissue, including, but not limited to, asthma, atherosclerosis, autoimmune disease, autoinflammatory disease, cancer, celiac disease, chronic prostatitis, colitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, lichen planus, mast cell activation syndrome, mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection, and vasculitis.

[0069] As used herein, "gastrointestinal condition" refers to any condition or disorder associated with any tissue of the gastrointestinal system. Gastrointestinal conditions include, but are not limited to, immune-related conditions of the gastrointestinal system, inflammatory conditions of the gastrointestinal system, microbial infection of gastrointestinal tissues, and conditions caused by eating disorders, metabolic disorders or metabolic deficiencies. Gastrointestinal conditions include, but are not limited to, inflammatory bowel disease, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, and cancer (e.g., colon cancer, rectal cancer, gastric cancer).

[0070] The term "treatment" or "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) undesirable physiological changes or disorders, such as the progression of multiple sclerosis, arthritis, or cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in disease extent, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or remission of disease symptoms, and remission (local or global). "Treatment" also means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those susceptible to or in whom the condition or disorder is to be prevented, as well as those already with the condition or disorder.

[0071] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject for whom a diagnosis, prognosis, or therapy is sought. Mammalian subjects include humans, or domestic, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, and the like.

[0072] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example an amount sufficient to modulate intracellular protein aggregation.

[0073] The term "therapeutically effective amount" is an amount effective for ameliorating symptoms of a disease. Since prevention can be considered a therapy, a therapeutically effective amount can also be a "prophylactically effective amount."

[0074] The term "recombinant human γδ TCR protein" or derivatives of this term referred to throughout the specification refers to any recombinant protein produced by standard genetic engineering methodologies from human γδ TCR sequences or functional derivatives or homologues thereof. Such recombinant proteins may also contain additional fusion elements such as dimerization domains. Non-limiting examples include fusions to support correct folding of the TCR, fusions to act as transmembrane domains (e.g. to support correct presentation of the TCR at the surface of a cell membrane), or fusions to extend half-life or increase size (e.g. human serum albumin fusion domains) or payload fusions as described elsewhere herein. These additional fusion elements may be produced with sequences derived from γδ TCR sequences or derivatives and homologues thereof, or alternatively, the fusion sequences may be non-TCR in origin.

[0075] The term "recombinant γδ TCR sequences" or "recombinant human TCR library" or "recombinant human TCR panel" or derivative terms derived therefrom referred to throughout the specification refers to a collection of more than one recombinant human γδ TCR protein, or a collection of more than two, or three, or four, or five, or ten recombinant human γδ TCR proteins. The collection may comprise a collection of sequences differing in at least one or more amino acids. The collection of recombinant TCRs may be displayed in a soluble form. Alternatively, for display purposes, the collection may also be bound, or fused, or tethered to inorganic or organic materials (non-limiting examples include "beads" or "plates" or "columns" or "phages"). Alternatively, such collections may also be displayed or displayed on a membrane or collections of membranes, such as those found in intact or living cells, or non-living membranes such as micelles. When expressing and displaying such a collection on one or more living cells, typically a collection of cognate expression vectors is also generated. This collection of cognate expression vectors can be used to initially engineer one or more cells to display the recombinant human γδ TCR protein, or a library or panel or collection of said proteins, to generate a library of cells expressing said TCR protein.

[0076] The terms "cognate binding partner" or "cognate binding partner candidate" or derivatives thereof referred to throughout this specification refer to proteins or derivatives thereof that have been identified as binding to recombinant human γδ TCR protein in a sequence-specific manner. For the discovery or screening or validation of such cognate binding partners, they can be presented or displayed in their natural environment (e.g., on the surface of one or more cells). They can also be presented or displayed as extracts or secretions from such cells, purified derivatives therefrom, or in recombinant form.

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

[0078] In this disclosure, the words "comprises," "comprising," "containing," "having," "includes," "including," and linguistic variations thereof have the meaning set forth in U.S. patent law and permit the presence of additional elements beyond those explicitly recited.

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

[0080] As used herein, the term "or" is understood to be inclusive unless otherwise specified or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural, unless otherwise specified or clear from context.

[0081] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0082] 6.3. Protein Constructs Containing BTNL3 / 8 Targeting Moieties In a first aspect, a protein construct is provided, the protein construct comprising a BTNL3 / 8 targeting moiety, a payload, and an optional linker connecting the targeting moiety to the payload.

[0083] 6.3.1.1 BTNL3 / 8-targeting components The BTNL3 / 8 targeting moiety specifically binds to human BTNL3, human BTNL8 and / or human BTNL3 / 8 heterodimer. In one embodiment, the BTNL3 / 8 targeting moiety is at least a portion of a T cell receptor (TCR) Vγ domain polypeptide, as described in more detail below. In another embodiment, the BTNL3 / 8 targeting moiety is an antigen-binding site of an antibody. In one embodiment, the BTNL3 / 8 targeting moiety inhibits or partially inhibits a function of BTNL3 / 8 when the BTNL3 / 8 targeting moiety binds to BTNL3 / 8. In one embodiment, the BTNL3 / 8 targeting moiety stimulates or activates a function of BTNL3 / 8 when the BTNL3 / 8 targeting moiety binds to BTNL3 / 8. In some embodiments, the BTNL3 / 8 targeting moiety inhibits or partially inhibits BTNL3 / 8 heterodimer function when the BTNL3 / 8 targeting moiety binds to the BTNL3 / 8 heterodimer, hi some embodiments, the BTNL3 / 8 targeting moiety stimulates or activates BTNL3 / 8 heterodimer function when the BTNL3 / 8 targeting moiety binds to the BTNL3 / 8 heterodimer.

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

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

[0086] In certain embodiments, the remaining residues of the Vγ domain CDR4 are independently selected at each position from the corresponding residues of human Vγ4, human Vγ2, or mouse Vγ7. In some embodiments, all of the remaining residues of the Vγ domain CDR4 are selected from the corresponding residues of human Vγ4, human Vγ2, or mouse Vγ7. In one embodiment, all of the remaining residues of the Vγ CDR4 are selected from the corresponding residues of human Vγ4. In one embodiment, all of the remaining residues of the Vγ CDR4 are selected from the corresponding residues of human Vγ2. In one embodiment, all of the remaining residues of the Vγ CDR4 are selected from the corresponding residues of mouse Vγ7. In one embodiment, the amino acid sequence of positions 87-90 of the Vγ domain is SEQ ID NO:1. In one embodiment, the amino acid sequence of positions 87-90 of the Vγ domain is SEQ ID NO: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. In one embodiment, the Vγ domain is a human Vγ4 domain. In one embodiment, the Vγ domain is a human Vγ2 domain, in which the amino acid in CDR4 is substituted at amino acid position 87 with aspartic acid or histidine and at amino acid position 90 with glycine or glutamic acid.

[0088] In one embodiment, the Vγ domain is human Vγ3 or human Vγ5. In a particular embodiment, the Vγ domain is human Vγ3 or human Vγ5, in which the amino acid in CDR4 is substituted with aspartic acid or histidine at amino acid sequence position 87 and with glycine or glutamic acid at amino acid sequence position 90. In one embodiment, the Vγ domain is a human Vγ domain having at least 70% sequence identity with human Vγ4.

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

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

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

[0092] (6.3.1.1.1 Pair with Vδ) In one embodiment, the BTNL3 / 8 targeting component of the protein construct does not further comprise a paired Vδ domain. In one embodiment, the BTNL3 / 8 targeting component of the protein construct comprises a Vδ domain paired with at least one additional Vγ domain. In one embodiment, the BTNL3 / 8 targeting component is a Vγ4 homodimer. In one embodiment, the BTNL3 / 8 targeting component of the protein construct further comprises a paired Vδ domain. In one embodiment, the Vδ domain is a human Vδ domain. In one embodiment, the human Vδ domain is Vδ1, Vδ2, Vδ3, Vδ5, or Vδ8. In one embodiment, the human Vδ domain is Vδ1. In one embodiment, the Vδ domain is a non-human mammalian Vδ domain.

[0093] ((a) Heterodimer format) In some embodiments, the Vδ domain is paired by heterodimerization of a first and a second polypeptide, one of which comprises a Vγ domain and one of which comprises a Vδ domain.

[0094] Heterodimeric interactions can include covalent and / or non-covalent interactions between multiple polypeptides comprising Vγ and Vδ domains. In a typical embodiment, the Vγ and Vδ domains are paired by orthogonal features that make homodimers less likely than heterodimers.

[0095] In some embodiments, the polypeptides each comprising a Vγ domain and a Vδ domain are covalently linked by at least one engineered disulfide bridge. The engineered disulfide bridge is an amino acid sequence that provides a non-endogenous cysteine ​​amino acid to two or more domains such that a non-native disulfide bond is formed when the two or more domains associate. In some of these embodiments, at least one disulfide bridge is engineered into the Vγ domain and the Vδ domain. In some embodiments, at least one disulfide bridge is engineered into a domain outside the variable region, such as into a constant region fused in frame with the variable region.

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

[0097] In one embodiment, one or more polypeptides of the paired Vγ / Vδ heterodimer further comprises a T cell receptor constant region. In one embodiment, the first T cell receptor constant region is fused in-frame to the C-terminus of the paired Vγ domain. In one embodiment, the first T cell receptor constant region is a human TCR constant region. In one embodiment, the first T cell receptor constant region is a human TCR β constant region. In one embodiment, the first T cell receptor constant region is a human TCR α constant region. In one embodiment, the first T cell receptor constant region is a human TCR γ constant region. In one embodiment, the paired Vγ / Vδ heterodimer further comprises a second T cell receptor constant region, which second T cell receptor constant region is fused in-frame to the C-terminus of the paired Vδ domain. In one embodiment, the second T cell receptor constant region is a human TCR α constant region. In one embodiment, the second T cell receptor constant region is a human TCR β constant region. In one embodiment, the second T cell receptor constant region is a human TCR δ constant region.

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

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

[0100] In some embodiments, the BTNL3 / 8 targeting moiety comprises more than one Vγ domain and / or Vδ domain. In some embodiments, the more than one Vγ domain and / or Vδ domain is multimerized. In some embodiments, all or part of the more than one Vγ domain and / or Vδ domain is fused in-frame to a T cell receptor constant region. In some embodiments, the more than one multimerized Vγ domain and / or Vδ domain comprises one or more internal linkers.

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

[0102] (b) Single-stranded in-frame fusion In some embodiments, the BTNL3 / 8 targeting moiety comprises a single-chain in-frame fusion of a Vγ domain and a paired Vδ domain. In some embodiments, the Vγ domain is N-terminal to the Vδ domain. In some embodiments, the Vγ domain is C-terminal to the Vδ domain. In various embodiments, the single-chain in-frame fusion of the Vγ domain and the Vδ domain comprises an internal linker sequence.

[0103] In some embodiments, the V5 domain is a human V5 domain. In one embodiment, the human V5 domain is V51, V52 or V55. In one embodiment, the human V5 domain is V51. In one embodiment, the single chain in-frame fusion further comprises at least one T cell receptor constant region. In one embodiment, the single chain in-frame fusion further comprises a first T cell receptor constant region, which is fused in-frame to the C-terminus of the Vgamma domain. In one embodiment, the first T cell receptor constant region is a human T cell receptor constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor beta constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor alpha constant region. In one embodiment, the first T cell receptor constant region is a human T cell receptor gamma constant region. In one embodiment, the single chain in-frame fusion further comprises a second T cell receptor constant region, which is fused in-frame to the C-terminus of the paired V5 domain. In one embodiment, the second T cell receptor constant region is a human T cell receptor alpha constant region. In one embodiment, the second T cell receptor constant region is a human T cell receptor beta constant region. In one embodiment, the second T cell receptor constant region is a human T cell receptor delta constant region. In one embodiment, the in-frame fusion of the V5 domain and the second T cell receptor constant region comprises an internal linker sequence between the V5 domain and the second T cell receptor constant region.

[0104] In some embodiments, the single chain in-frame fusion comprises more than one Vγ domain and / or Vδ domain. In some embodiments, the more than one Vγ domain and / or Vδ domain is multimerized. In some embodiments, the more than one multimerized Vγ domain and / or Vδ domain comprises one or more internal linkers. In some embodiments, all or part of the more than one Vγ domain and / or Vδ domain is fused in-frame to at least one T cell receptor constant region.

[0105] 6.3.2. Antibody-Based Targeting Moieties In some embodiments, the BTNL3 / 8 targeting moiety comprises an antibody that specifically binds to human BTNL3, human BTNL8 and / or human BTNL3 / 8 heterodimer. In some embodiments, the antibody is a full-length antibody fragment or antibody format, including but not limited to Fab fragments, Fv, scFv, tandem scFv, diabody, sc diabody, DART, tandem diabody (tandAb), minibody, camelid VHH, and other antibody fragments or formats known to those of skill in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al., MABS, 2017, Vol. 9, No. 2, 182-212, the teachings of which are incorporated herein by reference in their entirety.

[0106] In one embodiment, the antibody comprises an Fc domain capable of interacting with an Fc receptor. In one embodiment, the antibody comprises an Fc domain incapable of interacting with an Fc receptor. In some embodiments, the antibody has one or more engineered mutations in the amino acid sequence of the antibody domain that reduce effector functions that are naturally associated with the antibody binding. Effector functions include cellular functions that result from Fc receptors binding to the Fc portion of an antibody, such as, but not limited to, antibody-dependent cellular cytotoxicity (ADCC, also called antibody-dependent cell-mediated cytotoxicity), complement fixation (e.g., C1q binding), antibody-dependent cellular phagocytosis (ADCP), and opsonization. Engineered mutations that reduce effector function are described in detail in U.S. Patent Publication No. 2017 / 0137530, Armour et al., Eur. J. Immunol. 29(8)(1999)2613-2624, Shields et al., J. Biol. Chem. 276(9)(2001)6591-6604, and Oganesyan et al., Acta Cristallographica D64(2008)700-704, which are incorporated herein by reference in their entireties. In particular embodiments, the antibody has one or more engineered mutations in the amino acid sequence of the antibody domain that reduce binding of the Fc portion of the ROR binding molecule by the FcR receptor. In some embodiments, the FcR receptor is an FcRγ receptor. In particular embodiments, the FcR receptor is an FcγRIIa and / or FcγRIIIA receptor. In a particular embodiment, the one or more engineered mutations that reduce effector function are mutations in the CH2 domain of the antibody.

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

[0108] In various embodiments, the payload can include nucleotides, further nucleotides that contain a detectable moiety or toxin or that inhibit transcription, DNA, nucleic acids such as mRNA molecules that encode polypeptides such as enzymes, other RNA molecules (e.g., RNAi, miRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, and lncRNA), amino acids (e.g., amino acids that contain a detectable moiety or toxin or that inhibit translation), polypeptides (e.g., enzymes, biologics), lipids, carbohydrates, small molecules (e.g., small molecule drugs and small molecule toxins), and combinations thereof.

[0109] In some embodiments, the payload is a therapeutic agent, including, but not limited to, chemotherapeutic agents, immunomodulatory agents (e.g., cytokines, chemokines, or checkpoint inhibitors), hormones, and toxins (e.g., cytotoxic agents).

[0110] In one embodiment, the payload is an antibody. In one embodiment, the antibody comprises at least one antigen binding site (ABS) specific for the CD3 antigen. In one embodiment, the antibody comprises at least one ABS specific for the tumor necrosis factor alpha (TNFα) antigen. In one embodiment, the antibody comprises an Fc domain capable of interacting with an Fc receptor. In one embodiment, the antibody comprises an Fc domain incapable of interacting with an Fc receptor.

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

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

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

[0114] 6.3.1. Polypeptides In various embodiments, the payload is a polypeptide. In some embodiments, the payload is a polypeptide fused in-frame to the BTNL3 / 8 targeting moiety. In particular embodiments, the payload is fused in-frame to the C-terminus of the BTNL3 / 8 targeting moiety. In particular embodiments, the payload is fused in-frame to the N-terminus of the BTNL3 / 8 targeting moiety.

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

[0116] In some embodiments, the payload is an anti-inflammatory polypeptide. In particular embodiments, the anti-inflammatory polypeptide is an inhibitor of one or more proinflammatory cytokines. In particular embodiments, the anti-inflammatory polypeptide is an inhibitor of one or more of interleukin-1 (IL-1), IL-6, IL-12, IL-18, tumor necrosis factor alpha (TNFα), interferon gamma (INF-γ), or granulocyte-macrophage colony stimulating factor. In some embodiments, the anti-inflammatory polypeptide is a soluble cytokine receptor having anti-inflammatory activity, such as soluble tumor necrosis factor receptor p55, soluble tumor necrosis factor, p75, soluble IL-1 receptor type II, IL-18 binding protein, etc. In some embodiments, the anti-inflammatory polypeptide comprises an antibody antigen binding site that specifically binds to a proinflammatory cytokine.

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

[0118] (6.3.2.1.1 Antibody antigen binding site) In some embodiments, the payload is at least one antibody antigen-binding site (ABS). In some embodiments, the antibody antigen-binding site is configured as a Fab fragment, Fv, scFv, tandem scFv, diabody, sc diabody, DART, tandem diabody (tandAb), minibody, camelid VHH, nanobody, or other antibody fragment or format known to those of skill in the art. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al., MABS, 2017, Vol. 9, No. 2, 182-212, the teachings of which are incorporated herein by reference in their entirety.

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

[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-pro-inflammatory agent.

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

[0122] 6.3.2. Small Molecule Payloads In some embodiments, the payload is a small molecule. In some embodiments, the small molecule is a therapeutic agent (i.e., a small molecule drug). In some embodiments, the small molecule therapeutic agent is an immunomodulatory agent. In some 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 some embodiments, the small molecule therapeutic agent is a toxin.

[0123]

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

[0124]

[0102] Methods for preparing antibody drug conjugates (ADCs) that can be utilized to conjugate drugs to the protein constructs disclosed herein are described, for example, in U.S. Pat. No. 8,624,003 (pot process), U.S. Pat. No. 8,163,888 (one-step process), U.S. Pat. No. 5,208,020 (two-step process), U.S. Pat. No. 8,337,856, U.S. Pat. No. 5,773,001, U.S. Pat. No. 7,829,531, U.S. Pat. No. 5,208,020, U.S. Pat. No. 7,745,394, International Publication WO2017 / 136623, International Publication WO2017 / 015502, International Publication WO2017 / 015496, International Publication WO2017 / 015495, International Publication WO2004 / 010957, International Publication WO2005 / 077090, International Publication WO2005 / 082023, International Publication WO2006 / 0 65533, International Publication WO2007 / 030642, International Publication WO2007 / 103288, International Publication WO2013 / 173337, International Publication WO2015 / 057699, International Publication WO2015 / 095755, International Publication WO2015 / 123679, International Publication WO2015 / 157286, International Publication WO2017 / 165851, International Publication WO2009 / 073445, International Publication WO2010 / 06 8759, International Publication WO2010 / 138719, International Publication WO2012 / 171020, International Publication WO2014 / 008375, International Publication WO2014 / 093394, International Publication WO2014 / 093640, International Publication WO2014 / 160360, International Publication WO2015 / 054659, International Publication WO2015 / 195925, International Publication WO2017 / 160754, and Storz's literature (MAbs. 2015 Nov-Dec; 7(6): 989-1009), Lambert et al. (AdvTher, 2017 34: 1015), Diamantis et al. (British Journal of Cancer, 2016, 114, 362-367), Carrico et al. (Nat Chem Biol, 2007. 3: 321-2), We et al. (Proc Natl Acad Sci USA, 2009. 106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7:1052-67), Agarwal et al. (Proc Natl Acad Sci USA., 2013, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. (Drug Dev. and D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2014, 25:1331-41), Liang et al. (J Am Chem Soc., 2014, 136:10850-3), Drake et al., Curr Opin Chem Biol., 2015, 28:174-80, and York et al., BMC Biotechnology, 2016, 16(1):23, the teachings of each of which are incorporated herein by reference in their entireties.

[0125] 6.3.3. Nucleic Acid Payloads In some embodiments, the payload is a nucleic acid. The nucleic acid may be DNA or RNA, including, but not limited to, dsDNA, mRNA, miRNA, lncRNA, and siRNA, piRNA, snoRNA, snRNA, exRNA, and scaRNA.

[0126] (6.3.3.1 Optional Linker)

[0104] The protein constructs described herein optionally include a linker that connects the targeting moiety to the payload.

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

[0128] In some embodiments, the optional linker is a molecule conjugated to the targeting moiety. In various embodiments, the protein construct has modifications, including functional groups or chemically reactive groups, that can be used in downstream processes, such as attaching additional functional elements (e.g., payloads and BTNL3 / 8 targeting moieties) and downstream purification processes. In some embodiments, the linker comprises a cleavable molecule (e.g., a peptide that can be cleaved by a site-specific protease or other molecule that allows the linker to be cleaved into two or more fragments). In some embodiments, the modifications are chemically reactive groups, including, but not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), "click chemistry" groups (e.g., reactive alkyne groups), and aldehydes with formylglycine (FGly). In some embodiments, the modifications are functional groups, including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems, etc.). In some embodiments, the functional group or chemically reactive group comprises a cleavable peptide sequence. In particular embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions. In particular embodiments, the protease cleavage is performed by an intracellular protease. In particular embodiments, the protease cleavage is performed by an extracellular or membrane-bound protease. ADC therapy employing protease cleavage is described in detail in Choi et al., Theranostics, 2012; 2(2): 156-178., the teachings of which are incorporated herein by reference in their entirety.

[0129] In some embodiments, in addition to binding the targeting moiety to the payload, the linker can be used to allow site-specific conjugation of molecules to functional elements of the protein construct (e.g., the targeting moiety and the payload). In some embodiments, the linker can additionally be used to identify or detect the protein construct in vitro or in vivo. In some embodiments, the protein construct comprises more than one optional linker.

[0130] 6.4. Other Target Components In one aspect, described herein are panels or libraries of recombinant homodimeric and heterodimeric human γδ TCRs and methods of using said panels or libraries to identify specific cognate human binding domains or partners that determine or promote tissue-specific distribution of γδ T cells. These binding partners are often referred to as human native "self-antigens" and, until the discoveries described herein, have been very difficult / impossible to further determine or characterize. In one aspect of the invention, recombinant human γδ TCR proteins are described that exhibit tissue specificity or binding properties similar to those exhibited in their native cellular environment. In a further aspect of the invention, a method is described that includes (i) generating at least one recombinant γδ TCR, (ii) displaying or presenting or mixing said recombinant protein(s) to one or more potential cognate binding partners, preferably expressed on the cell surface, and (iii) then identifying or validating the specific γδ TCR / binding partner interaction. In one aspect of the invention, the resulting identified γδ TCR or sequences derived therefrom are employed as a targeting moiety to deliver a therapeutic payload to a target tissue or cell expressing the cognate binding partner. In a further embodiment, the cells expressing the identified cognate binding partner identified by the methods described herein are then targeted with another targeting moiety, such as an antibody or derivative thereof.

[0131] (6.5. How to Create)

[0109] The protein constructs described herein can be readily produced by protein expression using standard cell-free translation, transient transfection, and stable transfection approaches currently used for the production of T cell receptors or antibodies.

[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 expressed protein can be easily separated from undesired proteins and protein complexes using affinity resins (e.g., those that bind to affinity tags on the protein construct). Further purification can be performed using ion exchange chromatography, as is routinely used in the art.

[0133] Methods for evaluating the effectiveness and efficiency of the purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be evaluated by various criteria. Examples of criteria include, but are not limited to: 1) evaluating the percentage of total protein in the eluate obtained from the fully assembled protein construct, 2) evaluating the enrichment fold or increase of the method for purifying the desired product, e.g., comparing the total protein obtained from the fully assembled protein construct in the eluate with that of the starting sample, 3) evaluating the percentage of total protein or the decrease of undesired products, e.g., the incomplete complexes described above, e.g., determining the percentage or decrease of a particular undesired product (e.g., non-associated single polypeptide chains, any combination of polypeptide chain dimers, or any combination of polypeptide chain trimers).

[0134] 6.7. Pharmaceutical Compositions

[0112] In another aspect, there is provided a pharmaceutical composition comprising a protein construct comprising a BTNL3 / 8 targeting moiety and a payload as described herein, and a pharma- ceutically acceptable carrier or diluent. In a typical embodiment, the pharmaceutical composition is sterile. In one aspect, described herein is a pharmaceutical composition comprising any of the protein constructs described above and a pharma- ceutically acceptable carrier.

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

[0136] In various embodiments, the pharmaceutical composition comprises the protein construct in a concentration between 0.1 mg / ml and 100 mg / ml. In particular embodiments, the pharmaceutical composition comprises the protein construct in a concentration of 0.5 mg / ml, 1 mg / 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 comprises the protein construct in a concentration greater than 10 mg / ml. In some embodiments, the protein construct is present in a concentration of 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or even 50 mg / ml or higher. In particular embodiments, the protein construct is present in a concentration greater than 50 mg / ml.

[0137]

[0115] In various embodiments, the pharmaceutical compositions are described in detail in U.S. Patent No. 8,961,964, U.S. Patent No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, U.S. Patent Application No. 10 / 813,483, International Publication No. WO2014 / 066468, International Publication No. WO2011 / 104381, and International Publication No. WO2016 / 180941, the teachings of each of which are incorporated herein by reference in their entireties.

[0138] 6.8. Compositions for Use In one aspect, compositions for use are also provided. In one embodiment, compositions for use in therapy are provided, comprising a protein construct comprising a BTNL3 / 8 targeting moiety and a payload as described herein. The compositions can be used, for example, for the treatment of inflammatory conditions, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, metabolic disorders, cancer, immune-related disorders, autoimmunity, transplant rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke, and infectious diseases.

[0139]

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

[0140] (6.9. Treatment) In one aspect, a method of treatment is provided, comprising administering to a patient a protein construct comprising a BTNL3 / 8 targeting moiety and a payload, as described herein, in an effective amount to treat the patient. The protein construct of the present disclosure may be administered to a subject on its own or in the form of a pharmaceutical composition, for example, for the treatment of inflammatory conditions, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, metabolic disorders, cancer, immune-related disorders, autoimmunity, transplant rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke, and infectious diseases.

[0141] (6.9.1.1 Gastrointestinal symptoms)

[0119] In some aspects, described herein are methods for treating gastrointestinal conditions. Gastrointestinal conditions include, but are not limited to, immune-related conditions of the gastrointestinal system, inflammatory conditions of the gastrointestinal system, microbial infection of gastrointestinal tissue, and conditions caused by eating disorders, metabolic disorders or metabolic deficiencies. Gastrointestinal conditions include, but are not limited to, inflammatory bowel disease, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, and cancer (e.g., colon cancer, rectal cancer, gastric cancer). In some aspects, described herein are methods for treating a gastrointestinal condition in which gastrointestinal tissue expresses BTNL3 / 8, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to any one of claims 62-66 under conditions in which the gastrointestinal tissue expresses BTNL3 / 8. In one embodiment of this method, the payload of the protein construct is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an anti-inflammatory cytokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or transforming growth factor beta (TGFβ). 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. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload is an immunomodulatory agent.

[0142] (6.9.2.1 Inflammatory bowel disease) In one aspect, described herein is a method of treating inflammatory bowel disease comprising administering to a patient suffering from inflammatory bowel disease a therapeutically effective amount of any of the pharmaceutical compositions described above. In one embodiment, the inflammatory bowel disease is ulcerative colitis. In one embodiment, the inflammatory bowel disease is Crohn's disease. In one embodiment, the payload of the protein construct is an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent is an aminosalicylate. In one embodiment, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. In one embodiment, the anti-inflammatory agent is an anti-inflammatory cytokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22) or transforming growth factor beta (TGFβ). In one embodiment, the anti-inflammatory agent payload is an anti-pro-inflammatory agent. In one embodiment, the anti-inflammatory agent payload is a steroid. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the glucocorticoid is prednisone. In one embodiment, the glucocorticoid is hydrocortisone. In one embodiment, the payload of the protein construct is an antibiotic. In one embodiment, the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin or amoxicillin. In one embodiment, the payload of the protein construct is a calcineurin inhibitor. In one embodiment, the calcineurin inhibitor is cyclosporin A or tacrolimus. In one embodiment, the payload of the protein construct is an immunomodulator. In one embodiment, the immunomodulator is an immunosuppressant. In one embodiment, the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate or a thiopurine. In one embodiment, the payload of the protein construct is a protein payload. In one embodiment, the protein payload is an antibody, an antibody fragment or a single chain variable fragment. In one embodiment, the protein payload comprises and at least one ABS specific for the TNFα antigen. In one embodiment, the protein payload comprises the complementarity determining regions (CDRs) of adalimumab, infliximab, or certolizumab.In one embodiment, the protein payload comprises at least one ABS specific for an interleukin antigen. In one embodiment, the interleukin is IL-12, IL-23, or a combination thereof. In one embodiment, the protein payload comprises the CDRs of ustekinumab or brikinumab. In one embodiment, the biologic payload comprises at least one ABS specific for an integrin antigen. In one embodiment, the integrin is alpha4 integrin. In one embodiment, the protein payload comprises the CDRs of infliximab, natalizumab, or vedolizumab. In one embodiment, the protein construct comprises an analgesic payload. In one embodiment, the protein construct comprises a nutraceutical payload.

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

[0144] 6.9.4.1 Metabolic Disorder or Deficiency In some aspects, described herein is a method for treating a metabolic disorder or deficiency, comprising administering to a patient suffering from a metabolic disorder or deficiency a therapeutically effective amount of any of the pharmaceutical compositions described above. In some embodiments, the payload is a nutritional supplement. In some embodiments, the nutritional supplement is an enzyme or a vitamin.

[0145] 6.9.5.1 Regulation of the immune system

[0123] In some aspects, described herein are methods of regulating the immune system comprising administering a therapeutically effective amount of any of the pharmaceutical compositions described above to a patient having an immune-related condition. In some embodiments, the payload is an immunosuppressant. In some embodiments, the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine. In some embodiments, the payload is an immunostimulant. In some embodiments, the immunostimulant is a cytokine. EXAMPLES

[0146] (6.10. Example)

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

[0147] (6.10.1.1 method)

[0125] Non-limiting exemplary methods for designing and analyzing protein constructs containing BTNL3 / 8 targeting components containing TCR Vγ domains are described below. Methods for isolating primary lymphocytes, co-culturing with BTNL3 / 8-expressing HEK293 cells, and deep sequencing are also described in Di Marco Barros et al., Cell. 2016, (167), pp. 203-218.

[0148] (Human samples and primary lymphocyte isolation) Endoscopic biopsies were obtained from the ascending colon of adult donors undergoing routine colonoscopy. Primary intestinal lymphocytes were obtained by applying the method described by Clark et al., 2006, J. Invest. Dermatol. (126), pp. 1059-1070. Biopsies were washed for 20 min with 5 mL of washing medium (RPMI 1640 10% FCS, β-mercaptoethanol, penicillin [500 U / ml], streptomycin [500 mg / ml], metronidazole [5 mg / ml, Guy's Hospital, Department of Medicine], gentamicin [100 mg / ml, Sigma-Aldrich] and amphotericin 12.5 mg / ml [Thermo Fisher Scientific]). One endoscopic biopsy was placed on each matrix, which was then inverted and pressure was applied to press the biopsy into the matrix. Matrices were placed in 24-well plates (one per well) and covered with 2 mL RPMI 1640 (supplemented with 10% FCS, β-mercaptoethanol, penicillin [100 U / ml], streptomycin [100 mg / ml], metronidazole [1 mg / ml], gentamicin [20 mg / ml], amphotericin [2.5 mg / ml]), IL-2 (100 U / mL, Novartis Pharmaceutical UK) and IL-15 (10 ng / mL, Biolegend). 1 ml of medium was aspirated every other day (every 2nd day) and replaced with complete medium containing 2x concentrated cytokines. Cells were harvested and the remaining biopsies and empty wells were washed with PBS 0.02 mM HEPES. Cell suspensions were passed through a 70 mm nylon cell strainer, centrifuged at 400 g for 5 min, resuspended in complete medium without additional cytokines and immediately placed in co-cultures. Lymphocytes were used after 5-7 days in culture. PBMCs were isolated by Ficoll gradient from blood obtained from a blood donation service. (HEK293T co-culture assay) 5 × 10 transduced with either empty vector (EV), BTNL3, BTNL8 or BTNL3+8 5 10 x 10 HEK293T cells and 2 x 10 freshly harvested 5of primary human lymphocytes were co-cultured in 96-well plates in complete medium without supplemented cytokines and incubated at 37°C, 5% CO2 for 16 hours.

[0149] (Deep Sequencing) Amplification and sequencing of the mouse TRDV gene, TCRδ CDR3 from RNA purified from sorted Vγ7+ IELs, was performed using the Amp2Seq Platform (iRepertoire). Amplification and sequencing of the human TCRGVγ gene, TCRγ CDR3, was performed using the immunoSEQ Platform (Adaptive Biotechnologies).

[0150] (Design of soluble γδ TCR heterodimer)

[0128] The soluble γδTCR heterodimer containing the T cell receptor α and T cell receptor β constant regions used in the following examples was designed according to the literature by Xu et al., PNAS, 2011 Vol. 108; pp. 2414-2419.

[0151] 6.10.2.1 Example 1: γδ TCR variable regions isolated from intraepithelial lymphocytes from human intestinal tissue conferred induction of TCR activity on BTNL3 / 8

[0129] The TCR variable regions of human intestine-derived and BTNL3 / 8-responsive intraepithelial lymphocytes (IELs) were cloned and expressed in TCR-deficient cells (Figure 2). IELs were isolated from human intestinal tissue and co-cultured in HEK293T cells with simultaneous overexpression of BTNL3 / 8. Responsive IELs that showed TCR activation (high expression of CD25 and down-regulated Vγ) were then single-cell sorted. The variable regions of the γ and δ chains were amplified and cloned into lentiviral expression vectors (Figure 3A). TCR-deficient Jurkat cells (J76 cell line) were transduced and co-cultured with HEK293T cells expressing BTNL3 / 8 (Figure 3B). J76 cells were then sorted for TCR activation (CD69 expression and TCR down-regulation) (Figure 4A). Anti-CD3 antibody was used as a positive control for TCR activation. When co-cultured with BTNL3 / 8 expressing cells, J76 cells expressing a transduced TCR with Vγ4 and Vδ1 domains (H7 TCR) showed increased CD69 expression and downregulation of γδ TCR. Three independent J76 lines transduced with Vγ4Vδ1, B3, C11, and H7, representing three different CDR3 pairs obtained by the method shown in Figure 2, responded to BTNL3 / 8 expressing cells, but not the Vγ9Vδ2 line (Vγ9Vδ2) (Figure 4B). These results indicate that the Vγ4Vδ1 domain of human IELs is sufficient to confer TCR responsiveness to BTNL3 / 8.

[0152] 6.10.3.1 Example 2: CDR4 of Vγ4 is required for TCR responsiveness to BTNL3 / 8 To identify the Vγ4 region important for responsiveness to BTNL3 / 8, the entire Vγ4 domain of the H7-responsive TCR line was replaced with the Vγ2 region (FIG. 5). The fold change (FC) in CD69 expression % and percent TCR downregulation were determined in transduced J76 cells expressing Vγ4 TCR (H7 WT) or Vγ2-substituted TCRs when co-cultured with HEK293T expressing BTNL3 / 8. Replacement of the entire V region of the responsive Vγ4 H7 TCR with the Vγ2 coding sequence (Vγ2 H7), but not the CDR3 γ and entire δ chain, abolished TCR activation by BTNL3 / 8 expressing cells. However, the CDR1 of the responsive Vγ4 H7 TCR (H7 CDR1 Vγ2 ) and / or CDR2 (H7 CDR2 Vγ2 ) with Vγ2 coding sequences maintained TCR activation by BTNL3 / 8-expressing cells. These results indicate that CDR4 is required for TCR responses to BTNL3 / 8.

[0153] To further elucidate the region in Vγ4 that is essential for the response to BTNL3 / 8, two pairs of amino acids located in CDR4 were replaced with Vγ2 sequences (FIGS. 6 and 7). The fold change (FC) of CD69 expression % in transduced cells was determined when co-cultured with HEK293T expressing BTNL3 / 8, and the percent TCR downregulation was determined in J76 cells expressing Vγ4 TCR (H7 WT), Vγ2 TCR with CDR3 of H7 (Vγ2H7), and Vγ4 TCR with amino acid substitutions in CDR4 (FIG. 7). YA substitutions at amino acid positions 87 and 90 abolished TCR activation by BTNL3 / 8 expressing cells, whereas NL substitutions at amino acid positions 94 and 98 did not abolish TCR activation by BTNL3 / 8 expressing cells. These results confirm that amino acids 87 and 90 in the CDR4 region of Vγ4TCR are essential for TCR responsiveness to BTNL3 / 8.

[0154] 5.10.4.1 Example 3: Soluble TCR Vγ4 / Vδ heterodimers bind to BTNL3 / 8 expressing cells Soluble Vγ / Vδ TCR heterodimers were expressed and stabilized by leucine zipper complementation (FIG. 8). The Vγ or Vδ domains are fused in frame to TCRα or TCRβ constant regions lacking the transmembrane domain, followed by a leucine zipper sequence and a histidine tag / linker. Vγ4 / Vδ1 heterodimers correspond to SEQ ID NOs: 10 and 9. Vγ4 / Vδ2 heterodimers correspond to SEQ ID NOs: 10 and 11. Vγ2 / Vδ1 heterodimers correspond to SEQ ID NOs: 12 and 9. Vγ8 / Vδ1 heterodimers correspond to SEQ ID NOs: 13 and 9.

[0155] Soluble TCRs were used to stain HEK293T cells transduced with Flag-BTNL3+HA-BTNL8 or empty vector (Figure 10). Vγ4 / Vδ1 soluble TCR and Vγ4 / Vδ2 soluble TCR show strong binding to cell lines expressing BTNL3+BTNL8 but not to empty vector (EV) control cell lines. The results show that soluble TCRs expressing Vγ4 / Vδ1 domains or Vγ4 / Vδ2 domains bind to BTNL3 / 8 expressing cells but not to cells lacking BTNL3 / 8. Taken together, the results indicate that Vγ4 CDR4 is essential for BTNL3 / 8 induced TCR responses and also suggest that Vγ4 CDR4 interacts with BTNL3 / 8.

[0156] Soluble Vγ4 in cells expressing BTNL3+8 +To further evaluate the binding of the TCR constructs, HEK293T cells were transduced with pCSIGPW encoding the indicated BTNL3 and BTNL8 constructs or an empty vector (EV). Cells were then stained with soluble His-tagged Vγ4δ2 TCR for 45 min at 4°C, washed twice, stained with APC anti-His tag antibody (α-His) for 45 min at 4°C, washed twice again, and then analyzed by flow cytometry (Figure 9A). The cell populations shown in Figure 9B were stained in parallel with anti-FLAG and anti-HA antibodies to confirm that the lack of soluble TCR binding was not due to a failure to express the BTNL3+8 construct. This result demonstrates the ability of the soluble TCR construct to bind cells expressing BTNL3+BTNL8, and that this soluble TCR construct binds to Vγ4 + IgV domain mutants (e.g., L3) that have been previously described as unable to induce T cell responses GQFSS , L3 RI , L3 YQKAI ) did not bind to any of the antibodies. See Melandri et al., Nat. Immunol. 2018, which is incorporated by reference in its entirety.

[0157] 6.10.5.1 Example 4: Soluble TCRs that bind to BTNL3 / 8 expressing cells are internalized To determine whether soluble TCR bound to the surface of cells expressing BTNL3+BTNL8 is internalized, HEK293T cells were transduced to express wild type BTNL3 and BTNL8 (293T.L3L8) and stained with soluble His-tagged Vγ4Vδ2 TCR for up to 120 min at 37°C (Figure 11B). 293T.L3L8 cells were stained with soluble His-tagged Vγ4Vδ2 TCR for 120 min at 4°C to serve as a control where internalization was blocked at low temperature (Figure 11A). Cells were then stained with APC α-His tag antibody (α-His) for 45 min at 4°C. This shows a decrease in APC signal over time in the cell population incubated at 37°C, indicating that the cells rapidly internalize the soluble TCR construct (Figure 12).

[0158] To determine whether soluble TCR internalization is specific or the result of rapid cycling of cell surface BTNL molecules, the experiment was repeated and soluble TCR was compared to anti-BTNL3 antibody (rabbit polyclonal, Aviva Biosystems) (references included). HEK293T cells were transduced with an empty vector (293T.EV) and used as a negative control for staining with α-BTNL3. HEK293T cells were transduced with the BTNL3 construct L3 RI L8(293T.L3 RI L8) were used as a negative control for staining with soluble TCR. Results showed that α-BTNL3 staining was identical at 4°C and 37°C, indicating that α-BTNL3 specifically binds to cells expressing BTNL3 / 8, while the antibody remains on the surface of the cells (Figure 13A). Quantification of the results is shown in Figure 13B.

[0159] 6.10.6.1 Example 5: Soluble TCRs deliver payload to BTNL3 / 8 expressing cells To determine whether a payload can be delivered intracellularly via binding of soluble TCR to BTNL3 / 8 expressing cells, 293T.L3L8 or 293T.L3 RI L8 cells were incubated with soluble TCR pre-labeled on the carboxy terminus of the soluble TCR construct with APCα-His tag antibody. The complex was incubated at either 3 μg / mL or 10 μg / mL for 1 hour at either 4° C. or 37° C. The cells were then washed and treated with trypsin or DMEM (control) for 15 minutes as shown in FIG. 14. The results showed that the fraction of soluble TCR+α-His signal (APC fluorescence) was greater after trypsin treatment when cells were incubated with the complex at 37° C., protecting them from trypsin by internalization of the complex, compared to incubation at 4° C. (FIG. 15A). Thus, the results indicate that internalization of soluble TCR can be used as a means to deliver payloads intracellularly to BTNL3 / 8 expressing cells. Quantification of the results is shown in FIG. 15B.

[0160] Imaging cytometry was used to visualize intracellular delivery of APCα-His antibody payload via soluble TCR. RI 293T.L8 or 293T.L3L8 cells were incubated with soluble TCR+α-His antibody complexes for 1 hour at 4°C or 37°C. Then, treated with DMEM or trypsin (as above), cells were fixed and permeabilized (Figure 16A) or fixed and permeabilized and stained with the late endosome marker CD107a (Figures 16B and 16C). Imaging cytometry of negative controls allowed assessment of background APC signal (Figure 16A). Imaging cytometry results of cells incubated with TCR+α-His antibody complexes showed that when the complexes were incubated with cells at 4°C, after DMEM treatment the complexes were visualized around the cells, indicating that the majority of the complexes were bound to the cell surface, whereas after trypsinization the signal was completely lost (Figure 16B). However, complexes incubated with cells at 37°C still showed CD107a after trypsinization. + It is detected in the intracellular region proximal to the compartment (Figure 16C).

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

[0162] (7. Incorporation by Citation) All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0163] (8. Equivalents) While various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the invention. Many variations of the invention will be apparent to those skilled in the art upon review of this specification. The present application provides the following aspects of the invention. (Aspect 1) Components targeting BTNL3 / 8; payload; and An optional linker that connects the targeting moiety to the payload. A protein construct comprising: (Aspect 2) the BTNL3 / 8 targeting component comprises a Vγ domain; the amino acid at position 87 of the Vγ domain is aspartic acid or histidine, and the amino acid at position 90 of the Vγ domain is glycine or glutamic acid; and 2. The protein construct of embodiment 1, wherein the remaining residues of said Vγ CDR4 are, at each position, independently selected from the corresponding residues of a human or murine Vγ domain. (Aspect 3) 3. The protein construct of embodiment 2, wherein the remaining residues of said Vγ domain CDR4 are independently selected, at each residue position, from the corresponding residues of human Vγ4, human Vγ2, or mouse Vγ7. (Aspect 4) 3. The protein construct according to aspect 2, wherein the amino acid sequence at positions 87 to 90 of the Vγ domain is SEQ ID NO:1. (Aspect 5) 3. The protein construct according to aspect 2, wherein the amino acid sequence at positions 87 to 90 of the Vγ domain is SEQ ID NO:2. (Aspect 6) 4. The protein construct of embodiment 3, wherein all remaining residues of said Vγ domain CDR4 are selected from the corresponding residues of human Vγ4, human Vγ2, or mouse Vγ7. (Aspect 7) 7. The protein construct of embodiment 6, wherein the remaining residues of said Vγ CDR4 are selected from the corresponding residues of human Vγ4. (Aspect 8) 7. The protein construct of embodiment 6, wherein the remaining residues of said Vγ CDR4 are selected from the corresponding residues of human Vγ2. (Aspect 9) 7. The protein construct of embodiment 6, wherein the remaining residues of said Vγ CDR4 are selected from the corresponding residues of mouse Vγ7. (Aspect 10) 3. The protein construct of embodiment 2, wherein the Vγ domain is a human Vγ2 domain, in which the amino acid in CDR4 is substituted at amino acid position 87 with aspartic acid or histidine and at amino acid position 90 with glycine or glutamic acid. (Aspect 11) 3. The protein construct of embodiment 2, wherein said Vγ domain is a human Vγ4 domain. (Aspect 12) 12. The protein construct according to any one of aspects 1 to 11, wherein the Vγ domain CDR3 is a human or mouse Vγ CDR3 sequence. (Aspect 13) 13. The protein construct of embodiment 12, wherein said Vγ domain CDR3 comprises a human Vγ4 CDR3 sequence. (Aspect 14) 13. The protein construct of embodiment 12, wherein said Vγ domain CDR3 comprises a human Vγ2 CDR3 sequence. (Aspect 15) 13. The protein construct of embodiment 12, wherein said Vγ domain CDR3 comprises a mouse Vγ7 CDR3 sequence. (Aspect 16) 16. The protein construct according to any one of embodiments 2 to 15, wherein the J region is a Vγ J region. (Aspect 17) 17. The protein construct of embodiment 16, wherein said J region is a human Vγ J region. (Aspect 18) 17. The protein construct of embodiment 16, wherein said J region is a mouse Vγ J region. (Aspect 19) 17. The protein construct according to embodiment 16, wherein the J region comprises a SEQ ID NO: selected from the group consisting of SEQ ID NOs: 15 to 18. (Aspect 20) 20. The protein construct of any one of embodiments 1 to 19, wherein the BTNL3 / 8 targeting component further comprises a paired Vδ domain. (Aspect 21) 21. The protein construct of embodiment 20, wherein the Vγ and Vδ domains are covalently linked by at least one disulfide bond. (Aspect 22) 22. The protein construct of embodiment 21, wherein the Vγ and Vδ domains are paired through a specific heterodimeric interaction. (Aspect 23) 23. The protein construct of embodiment 22, wherein said heterodimeric interaction is leucine zipper complementarity. (Aspect 24) 24. The protein construct of embodiment 23, wherein said targeting moiety comprises SEQ ID NO:9. (Aspect 25) 24. The protein construct of embodiment 23, wherein said targeting moiety is SEQ ID NO:10. (Aspect 26) 24. The protein construct of embodiment 23, wherein said targeting moiety is SEQ ID NO:11. (Aspect 27) 21. The protein construct of embodiment 20, wherein said targeting moiety comprises a single-chain in-frame fusion of a Vγ domain and a Vδ domain. (Aspect 28) 28. The protein construct of embodiment 27, wherein the Vγ domain is N-terminal to the Vδ domain. (Aspect 29) 28. The protein construct of embodiment 27, wherein the Vγ domain is C-terminal to the Vδ domain. (Aspect 30) 30. The protein construct according to any one of aspects 27 to 29, wherein the single-chain in-frame fusion of the Vγ and Vδ domains comprises an internal linker sequence. (Aspect 31) The protein construct according to any one of aspects 20 to 24 or 27 to 30, wherein the Vδ domain is a human Vδ domain. (Aspect 32) 32. The protein construct of embodiment 31, wherein the human V5 domain is V51, V52 or V55. (Aspect 33) 33. The protein construct of embodiment 32, wherein the human V5 domain is V51. (Aspect 34) 34. The protein construct according to any one of aspects 2 to 33, further comprising a first T cell receptor constant region, wherein the first T cell receptor constant region is fused in-frame to the C-terminus of the Vγ domain. (Aspect 35) 35. The protein construct of embodiment 34, wherein said first T cell receptor constant region is a human T cell receptor constant region. (Aspect 36) 36. The protein construct of embodiment 35, wherein said first T cell receptor constant region is a human T cell receptor beta constant region. (Aspect 37) 36. The protein construct of embodiment 35, wherein said first T cell receptor constant region is a human T cell receptor alpha constant region. (Aspect 38) 36. The protein construct of embodiment 35, wherein said first T cell receptor constant region is a human T cell receptor gamma constant region. (Aspect 39) 39. The protein construct according to any one of aspects 20 to 38, wherein the targeting moiety further comprises a second T cell receptor constant region, wherein the second T cell receptor constant region is fused in-frame to the C-terminus of the paired Vδ domain. (Aspect 40) 40. The protein construct of embodiment 39, wherein said second T cell receptor constant region is a human T cell receptor alpha constant region. (Aspect 41) 40. The protein construct of embodiment 39, wherein said second T cell receptor constant region is a human T cell receptor beta constant region. (Aspect 42) 40. The protein construct of embodiment 39, wherein said second T cell receptor constant region is a human T cell receptor delta constant region. (Aspect 43) 43. The protein construct of any one of aspects 39 to 42, wherein the in-frame fusion of the Vδ domain and the second T cell receptor constant region comprises an internal linker sequence between the Vδ domain and the second T cell receptor constant region. (Aspect 44) 44. The protein construct according to any one of embodiments 1 to 43, wherein said payload is a protein payload fused in-frame to the targeting moiety. (Aspect 45) 45. The protein construct of embodiment 44, wherein the payload is a polypeptide. (Aspect 46) 46. ​​The protein construct of embodiment 45, wherein the payload is a peptide. (Aspect 47) 46. ​​The protein construct of embodiment 45, wherein said payload is a cytokine. (Aspect 48) 46. ​​The protein construct according to embodiment 45, wherein the payload is an antibody. (Aspect 49) 49. The protein construct according to embodiment 48, wherein said payload is a single chain variable fragment (scFv). (Aspect 50) 50. A protein construct according to any one of embodiments 47 to 49, wherein said antibody comprises at least one antigen binding site (ABS) specific for a cytokine antigen. (Aspect 51) 50. The protein construct according to any one of embodiments 47 to 49, wherein said antibody is at least one ABS specific for a tumor necrosis factor alpha (TNFα) antigen. (Aspect 52) 52. The protein construct according to any one of aspects 47 to 49 to 51, wherein the antibody comprises an Fc domain capable of interacting with an Fc receptor. (Aspect 53) 52. The protein construct according to any one of aspects 47 or 48 to 51, wherein the antibody comprises an Fc domain that is not capable of interacting with an Fc receptor. (Aspect 54) 43. The protein construct according to any one of embodiments 1 to 42, wherein said payload is a small molecule. (Aspect 55) 55. The protein construct of embodiment 54, wherein the payload is a hormone. (Aspect 56) 55. The protein construct of embodiment 54, wherein the payload is a nucleic acid. (Aspect 57) 57. The protein construct of embodiment 56, wherein the payload is an inhibitory RNA (RNAi). (Aspect 58) 58. The protein construct according to any one of embodiments 1 to 57, wherein said optional linker is a peptide fused in-frame to the targeting moiety. (Aspect 59) 59. The protein construct of embodiment 58, wherein said optional linker is fused in-frame to the C-terminus of the targeting moiety. (Aspect 60) 59. The protein construct of embodiment 58, wherein said optional linker is fused in-frame to the N-terminus of the targeting moiety. (Aspect 61) 58. The protein construct according to any one of embodiments 1 to 57, wherein said optional linker is a molecule conjugated to a targeting moiety. (Aspect 62) 62. A pharmaceutical composition comprising the protein construct according to any one of embodiments 1 to 61 and a pharma- ceutically acceptable carrier. (Aspect 63) 63. The pharmaceutical composition of embodiment 62, wherein the pharmaceutical composition is suitable for parenteral administration. (Aspect 64) The pharmaceutical composition of embodiment 63, wherein said administering is intravenous administration. (Aspect 65) The pharmaceutical composition of embodiment 63, wherein said administration is intramuscular administration. (Aspect 66) The pharmaceutical composition of embodiment 63, wherein said administering is subcutaneous. (Aspect 67) A method for treating a gastrointestinal condition in which gastrointestinal tissue expresses BTNL3 / 8, the method comprising administering a therapeutically effective amount of a pharmaceutical composition described in any one of aspects 62 to 66 to a patient suffering from the condition in which gastrointestinal tissue expresses BTNL3 / 8. (Aspect 68) The method of embodiment 67, wherein the payload of the protein construct is an anti-inflammatory agent. (Aspect 69) The method of embodiment 68, wherein the anti-inflammatory agent is an aminosalicylate. (Aspect 70) The method of embodiment 68, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent. (Aspect 71) 70. The method of embodiment 68, wherein the anti-inflammatory agent is an anti-inflammatory cytokine, optionally interleukin 10 (IL-10), interleukin 22 (IL-22), or transforming growth factor beta (TGFβ). (Aspect 72) The method of embodiment 68, wherein the anti-inflammatory agent is an anti-pro-inflammatory agent. (Aspect 73) The method of embodiment 68, wherein the anti-inflammatory agent is a steroid. (Aspect 74) The method of embodiment 73, wherein the steroid is a glucocorticoid. (Aspect 75) The method of embodiment 74, wherein said glucocorticoid is prednisone. (Aspect 76) The method of embodiment 74, wherein said glucocorticoid is hydrocortisone. (Aspect 77) The method of embodiment 67, wherein the payload is an immunomodulatory agent. (Aspect 78) A method for treating inflammatory bowel disease, comprising administering to a patient suffering from inflammatory bowel disease a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 62 to 66. (Aspect 79) The method of embodiment 78, wherein said inflammatory bowel disease is ulcerative colitis. (Aspect 80) The method of embodiment 78, wherein said inflammatory bowel disease is Crohn's disease. (Aspect 81) The method according to any one of aspects 78 to 80, wherein the payload of said protein construct is an anti-inflammatory agent. (Aspect 82) The method of embodiment 81, wherein the anti-inflammatory agent is an aminosalicylate. (Aspect 83) The method of embodiment 81, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. (Aspect 84) The method of embodiment 81, wherein the anti-inflammatory agent is an anti-inflammatory cytokine. (Aspect 85) The method of embodiment 81, wherein the anti-inflammatory payload is an anti-pro-inflammatory agent. (Aspect 86) The method of embodiment 81, wherein the anti-inflammatory payload is a steroid. (Aspect 87) The method of embodiment 86, wherein the steroid is a glucocorticoid. (Aspect 88) The method of embodiment 87, wherein said glucocorticoid is prednisone. (Aspect 89) The method of embodiment 87, wherein said glucocorticoid is hydrocortisone. (Aspect 90) The method according to any one of aspects 78 to 80, wherein the payload of the protein construct is an antibiotic. (Aspect 91) 91. The method of embodiment 90, wherein the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin. (Aspect 92) The method according to any one of aspects 78 to 80, wherein the payload of the protein construct is a calcineurin inhibitor. (Aspect 93) 93. The method of embodiment 92, wherein said calcineurin inhibitor is cyclosporin A or tacrolimus. (Aspect 94) The method according to any one of aspects 78 to 80, wherein the payload of said protein construct is an immunomodulatory agent. (Aspect 95) The method of embodiment 94, wherein the immunomodulatory agent is an immunosuppressant. (Aspect 96) 96. The method of embodiment 95, wherein the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine. (Aspect 97) The method according to any one of aspects 78 to 80, wherein the payload of said protein construct is a protein payload. (Aspect 98) 98. The method of embodiment 97, wherein the protein payload is an antibody, an antibody fragment, or a single chain variable fragment. (Aspect 99) 99. The method of embodiment 97 or 98, wherein said protein payload comprises and at least one ABS specific for a TNFα antigen. (Aspect 100) 100. The method of embodiment 99, wherein said protein payload comprises the complementarity determining regions (CDRs) of adalimumab, infliximab, or certolizumab. (Aspect 101) 99. The method of embodiment 97 or 98, wherein said protein payload comprises at least one ABS specific for an interleukin antigen. (Aspect 102) The method of embodiment 101, wherein said interleukin is IL-12, IL-23, or a combination thereof. (Aspect 103) The method of embodiment 102, wherein said protein payload comprises the CDRs of ustekinumab or brikinumab. (Aspect 104) The method of embodiment 97 or 98, wherein said biologic payload comprises at least one ABS specific for an integrin antigen. (Aspect 105) The method of embodiment 104, wherein the integrin is alpha4 integrin. (Aspect 106) The method of embodiment 105, wherein said protein payload comprises the CDRs of infliximab, natalizumab, or vedolizumab. (Aspect 107) The method according to any one of aspects 78 to 80, wherein said protein construct comprises an analgesic payload. (Aspect 108) The method of any one of embodiments 78 to 80, wherein said protein construct comprises a nutraceutical payload. (Aspect 109) A method for treating irritable bowel syndrome, comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 62 to 66 to a patient suffering from irritable bowel syndrome. (Aspect 110) A method for treating diverticulitis, comprising administering to a patient suffering from diverticulitis a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 1 to 66. (Aspect 111) 111. The method of embodiment 109 or 110, wherein the payload is an antibiotic. (Aspect 112) The method of embodiment 111, wherein the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin. (Aspect 113) A method for treating celiac disease, comprising administering to a patient suffering from celiac disease a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 62 to 66. (Aspect 114) The method of embodiment 113, wherein the payload is an immunosuppressant. (Aspect 115) The method of embodiment 114, wherein the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine. (Aspect 116) A method for treating a microbial infection comprising administering to a patient suffering from a microbial infection a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 62 to 66. (Aspect 117) The method of embodiment 116, wherein the payload is an antibacterial agent. (Aspect 118) The method of embodiment 117, wherein the antimicrobial agent is an antiparasitic agent, an antibiotic agent, an antifungal agent, or an antiviral agent. (Aspect 119) A method for treating a metabolic disorder or metabolic deficiency, comprising administering to a patient suffering from a metabolic disorder or metabolic deficiency a therapeutically effective amount of a pharmaceutical composition according to any one of aspects 62 to 66. (Aspect 120) The method of embodiment 119, wherein the payload is a dietary supplement. (Aspect 121) The method of embodiment 120, wherein the dietary supplement is an enzyme or a vitamin. (Aspect 122) A method for regulating the immune system, comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of aspects 62 to 66 to a patient suffering from an immune-related condition. (Aspect 123) The method of embodiment 122, wherein the payload is an immunosuppressant. (Aspect 124) The method of embodiment 123, wherein the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine. (Aspect 125) The method of embodiment 122, wherein the payload is an immunostimulant. (Aspect 126) The method of embodiment 125, wherein the immunostimulant is a cytokine. (Aspect 127) The method of any one of aspects 67 to 126, wherein at least a portion of the BTNL3 / 8 targeting moiety and / or payload is transported into the cell. (Aspect 128) The method of any one of aspects 67 to 126, wherein the BTNL3 / 8 targeting moiety and / or payload is not internalized within the cell. (Aspect 129) A recombinant γδ TCR protein comprising at least one sequence having at least 90% sequence identity with a sequence from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. (Aspect 130) 130. A recombinant γδ TCR protein according to embodiment 129, wherein the protein comprises two or more sequences having at least 90% sequence identity with a sequence from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. (Aspect 131) 131. The recombinant γδ TCR protein according to any one of aspects 129 to 130, wherein said protein comprises a sequence having at least 90% sequence identity with SEQ ID NO:10. (Aspect 132) 132. The recombinant γδ TCR protein according to any one of aspects 129 to 131, wherein the protein comprises a sequence having at least 90% sequence identity with SEQ ID NO:10 and SEQ ID NO:11. (Aspect 133) 132. The recombinant γδ TCR protein according to any one of aspects 129 to 131, wherein the protein comprises a sequence having at least 90% sequence identity with SEQ ID NO:9 and SEQ ID NO:10. (Aspect 134) The recombinant γδ TCR protein according to any one of aspects 129 to 133, wherein the protein comprises a sequence having at least 90% sequence identity to either SEQ ID NO: 9 without a His tag or SEQ ID NO: 11 without a His tag.

Claims

1. Components targeting BTNL3 / 8; payload; and An optional linker that connects the BTNL3 / 8 targeting moiety to the payload. A protein construct comprising: The BTNL3 / 8 targeting component comprises a T cell receptor (TCR) Vγ domain, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4; CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO:3, the CDR4 of the TCR Vγ domain is located between the CDR2 and CDR3 regions of the TCR Vγ domain; CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG, and The protein construct, wherein the payload is a protein payload or a small molecule fused in-frame to the BTNL3 / 8 targeting moiety.

2. The protein construct of claim 1, wherein the CDR3 of the TCR Vγ domain comprises a CDR3 of a human Vγ.

3. 2. The protein construct of claim 1, wherein the J region comprises an amino acid sequence set forth in a human Vγ J region, a mouse Vγ J region, or a sequence selected from SEQ ID NOs: 15-18.

4. The protein construct of any one of claims 1 to 3, wherein the BTNL3 / 8 targeting component further comprises a paired Vδ domain.

5. The protein construct of claim 4, wherein the Vγ domain and the Vδ domain are covalently linked by at least one disulfide bond.

6. The protein construct of claim 5, wherein the Vγ domain and the Vδ domain are paired through a specific heterodimeric interaction.

7. The protein construct of claim 6, wherein the heterodimeric interaction is leucine zipper complementarity.

8. The protein construct of claim 7, wherein the BTNL3 / 8 targeting component comprises the amino acid sequence set forth in SEQ ID NO:

10.

9. The protein construct of claim 4, wherein the BTNL3 / 8 targeting component comprises a single-chain in-frame fusion of the Vγ and Vδ domains.

10. The protein construct of claim 9, wherein the Vγ domain is N-terminal to the Vδ domain.

11. The protein construct of claim 9, wherein the Vγ domain is C-terminal to the Vδ domain.

12. The protein construct of any one of claims 9 to 11, wherein the single-chain in-frame fusion of the Vγ and Vδ domains comprises an internal linker sequence.

13. The protein construct of any one of claims 4 to 12, wherein the Vδ domain comprises a human Vδ domain selected from Vδ1, Vδ2 or Vδ5.

14. further comprising a first T cell receptor constant region, The protein construct of any one of claims 1 to 13, wherein the first T cell receptor constant region is fused in-frame to the C-terminus of the Vγ domain.

15. The protein construct of claim 14, wherein the first T cell receptor constant region is a human T cell receptor constant region.

16. The protein construct of claim 15, wherein the human T cell receptor constant region is a human T cell receptor beta constant region, a human T cell receptor alpha constant region, or a human T cell receptor gamma constant region.

17. The BTNL3 / 8 targeting component further comprises a second T cell receptor constant region; 17. The protein construct of any one of claims 14 to 16, wherein the second T cell receptor constant region is fused in-frame to the C-terminus of the paired Vδ domain.

18. The protein construct of claim 17, wherein the second T cell receptor constant region is a human T cell receptor alpha constant region, a human T cell receptor beta constant region, or a human T cell receptor delta constant region.

19. The protein construct of claim 17 or 18, 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.

20. The protein construct of any one of claims 1 to 19, wherein the payload comprises a polypeptide, a peptide, a cytokine, or an antibody.

21. The protein construct of any one of claims 1 to 19, wherein the small molecule comprises a hormone, a nucleic acid, or an inhibitory RNA (RNAi).

22. The protein construct of any one of claims 1 to 21, wherein the optional linker is a peptide fused in-frame to the BTNL3 / 8 targeting moiety or comprises a molecule conjugated to the BTNL3 / 8 targeting moiety.

23. A pharmaceutical composition comprising a protein construct according to any one of claims 1 to 22.

24. 24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is suitable for parenteral administration.

25. 25. The pharmaceutical composition of claim 24, wherein the parenteral administration comprises intravenous administration, intramuscular administration, or subcutaneous administration.

26. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises an anti-inflammatory agent.

27. 27. The pharmaceutical composition of claim 26, wherein the anti-inflammatory agent is an aminosalicylate, a nonsteroidal anti-inflammatory agent, an anti-inflammatory cytokine, an anti-pro-inflammatory agent, or a steroid.

28. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises an antibiotic.

29. 29. The pharmaceutical composition of claim 28, wherein the antibiotic payload is rifaximin, ciprofloxacin, metronidazole, moxifloxacin, or amoxicillin.

30. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises a calcineurin inhibitor.

31. 31. The pharmaceutical composition of claim 30, wherein the calcineurin inhibitor is cyclosporin A or tacrolimus.

32. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises an immunomodulatory agent.

33. 33. The pharmaceutical composition of claim 32, wherein the immunomodulatory agent is an immunosuppressant.

34. 34. The pharmaceutical composition of claim 33, wherein the immunosuppressant is azathioprine, 6-mercaptopurine, methotrexate, or a thiopurine.

35. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises an antibacterial agent.

36. 36. The pharmaceutical composition of claim 35, wherein the antibacterial agent is an antiparasitic agent, an antifungal agent, or an antiviral agent.

37. The pharmaceutical composition of any one of claims 23 to 25, wherein the payload comprises a nutritional supplement.

38. 38. The pharmaceutical composition of claim 37, wherein the dietary supplement is an enzyme or a vitamin.

39. A recombinant γδ TCR protein comprising at least one sequence having at least 90% sequence identity with SEQ ID NO: 10, The recombinant γδ TCR protein is capable of targeting BTNL3 / 8-expressing cells, the recombinant γδ TCR protein comprising a TCR Vγ domain that targets BTNL3 / 8, the TCR Vγ domain comprising a J region and complementarity determining regions CDR1, CDR2, CDR3, and CDR4, wherein CDR1 of the TCR Vγ domain comprises an amino acid sequence selected from EGSTGY or EGSNGY, CDR2 of the TCR Vγ domain comprises an amino acid sequence selected from YDSYTSSV or YDSYNSKV, and CDR3 of the TCR Vγ domain comprises an amino acid sequence selected from ATWDE or ATWDG, and CDR4 of the TCR Vγ domain comprises the amino acid sequence set forth in SEQ ID NO:

3.

40. The recombinant γδ TCR protein according to claim 39, wherein the recombinant γδ TCR protein comprises the sequence of SEQ ID NO: 10.

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