EX VIVO Gamma Delta T Cell Population

Abstract

This invention provides a method for obtaining a population of Vγ4 T cells for T-cell immunotherapy against cancer. [Solution] An ex vivo method for modulating Vγ4T cells is provided, comprising administering an antibody or fragment thereof that specifically binds to the gamma variable 4 (Vγ4) chain of the γδT cell receptor (TCR) and does not bind to the gamma variable 2 (Vγ2) chain of the γδTCR to a cell population including Vγ4T cells.

Description

[Technical Field]

[0001] This invention relates to a population of gamma delta T cells contacted with an anti-TCR gamma variable 4 (anti-Vγ4) antibody. [Background technology]

[0002] The growing interest in T-cell immunotherapy for cancer focuses on the apparent ability of subsets of CD8+ and CD4+ alpha-beta (αβ) T cells to recognize cancer cells and mediate host-protective functionality, particularly when suppression is released through clinically mediated antagonism of inhibitory pathways exerted by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC-restrictive, which can lead to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express distinct, typical γδ T cell receptors (TCRs) on their surface. These TCRs consist of one gamma (γ) chain and one delta (δ) chain, each undergoing chain rearrangement, but with a limited number of V genes compared to αβ T cells. The main TRVG gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9, as well as the non-functional genes TRGV10, TRGV11, TRGVA, and TRGVB. The most frequent TRDV gene segments encode Vδ1, Vδ2, and Vδ3, and also encode several V segments with both Vδ and Vα names (Adams et al., 296:30-40 (2015) Cell Immunol.). Human γδ T cells can be broadly classified based on their TCR chains. This is because specific γ and δ types are found more commonly in cells in one or more tissue types, though not exclusively. For example, γδ T cells, which are found in most blood, express Vδ2 TCR, commonly Vγ9Vδ2, which is less common in γδ T cells in tissues such as skin, which more frequently use Vδ1 TCR, which is paired with the γ chain, for example, often with Vγ4 in the gut.

[0004] However, to date, due to the high homology between Vγ4TCR and other TRGV family members such as Vγ2TCR, a modality capable of targeting only Vγ4TCR has been impossible. Therefore, there remains an unmet need for Vγ4-specific antibodies, including specific antibodies that specifically bind to or specifically modulate Vγ4TCR. [Overview of the Initiative]

[0005] According to a first aspect of the present invention, an ex vivo method for modulating gamma-variable 4 (Vγ4) T cells is provided, comprising administering an antibody or fragment thereof that specifically binds to the Vγ4 chain of the γδ T cell receptor (TCR) and does not bind to the gamma-variable 2 (Vγ2) chain of the γδ TCR to a cell population containing Vγ4 T cells. It should be understood that this relates to the Vγ4 chain and Vγ2 from the same species. Preferably, according to all aspects and embodiments described herein, the species is Homo sapiens (human), and therefore the present invention also provides an ex vivo method for modulating gamma-variable 4 (Vγ4) T cells, comprising administering an antibody or fragment thereof that specifically binds to the human gamma-variable 4 (Vγ4) chain of the γδ T cell receptor (TCR) and does not bind to the human gamma-variable 2 (Vγ2) chain of the γδ TCR. For example, the human Vγ4 chain may have the sequence of amino acids 1-99 of SEQ ID NO: 1, and / or the human Vγ2 chain may have the sequence of SEQ ID NO: 335. In other species, the antibody or fragment specifically binds to the species-specific ortholog of the human gamma variable 4 (Vγ4) chain of the γδ T cell receptor (TCR), and to the species-specific ortholog of the human gamma variable 2 (Vγ2) chain of the γδ TCR. It does not bind to. Therefore, the antibody or fragment may specifically bind to the human gamma variable 4 (Vγ4) chain of the γδ T cell receptor (TCR) or its non-human orthologue having a sequence corresponding to amino acids 1-99 of SEQ ID NO: 1, but may not bind to the human gamma variable 2 (Vγ2) chain of the γδ TCR or its non-human orthologue having a sequence corresponding to SEQ ID NO: 335. In this context, an orthologue may mean a gamma chain sequence that has the highest sequence similarity to the reference sequence, or preferably has the same function (e.g., interaction with orthologous congener ligands in vivo). For example, in mice, the protein designated as Vγ7 under Heilig & Tonegave nomenclature is functionally most closely related to human Vγ4 (Barros et al. (2016) Cell, 167:203-218.e17).

[0006] This represents a significant advance in this field. For example, in humans, the Vγ4 and Vγ2 chains are highly homologous (91% sequence identity), differing by only nine amino acids. Three of these nine mutations map across CDR1 and CDR2, while four of these nine mutations map to a sub-region of framework region 3 (FR3)—amino acids 67-82 of SEQ ID NO: 1. Due to the extremely high sequence similarity between the Vγ4 and Vγ2 chains, it was previously thought impossible to develop an antibody or fragment thereof that could specifically distinguish between human Vγ4 and human Vγ2 chains of γδTCR. Surprisingly and contrary to the common view in the art, the inventors were able to develop such an antibody using the method described in more detail herein. Thus, the present invention relates to an antibody and fragment thereof that can specifically modulate Vγ4-containing γδTCRs. This provides a method for conducting a biological study.

[0007] The antibodies or fragments described herein may bind to an epitope of the human Vγ4 chain of the γδTCR containing one or more amino acid residues within the amino acid region 67-82 of SEQ ID NO: 1.

[0008] According to a further embodiment of the present invention, CDR3 containing one of sequence numbers 2 to 47, preferably a sequence having at least 80% sequence identity with sequence number 10 and / or sequence number 33, CDR2 includes one of sequence numbers 48-70 and sequences A1-A23 (in Figure 1), preferably sequence number 56 and / or A9, having at least 80% sequence identity, and / or An anti-Vγ4 antibody or a fragment thereof, comprising one or more CDR1 sequences having at least 80% sequence identity with any one of SEQ ID NOs. 71 to 116, preferably SEQ ID NOs. 79 and / or SEQ ID NOs. 102, An ex vivo method for modulating gamma-variable 4(Vγ4)T cells is provided, which includes administering them to a cell population containing Vγ4T cells.

[0009] In some embodiments, the anti-Vγ4 antibody or a fragment thereof A heavy chain CDR3 (HCDR3) containing one of sequence numbers 2 to 24, preferably a sequence having at least 80% sequence identity with sequence number 10, A heavy chain CDR2 (HCDR2) containing one of sequence numbers 48 to 70, preferably a sequence having at least 80% sequence identity with sequence number 56, and / or It may contain one or more heavy chain CDR1 (HCDR1) sequences, preferably one of sequence numbers 71 to 93, which have at least 80% sequence identity with sequence number 79.

[0010] Alternatively, an anti-Vγ4 antibody or a fragment thereof may be used. A light chain CDR3 (LCDR3) containing one of sequence numbers 25 to 47, preferably a sequence having at least 80% sequence identity with sequence number 33, One of sequences A1 to A23 (in Figure 1), preferably sequence number A9 and at least 8 Light chain CDR2 (LCDR2) containing a sequence with 0% sequence identity, and / or It may contain one or more light chain CDR1 (LCDR1) sequences that have at least 80% sequence identity with any one of sequence numbers 94 to 116, preferably sequence number 102.

[0011] In some embodiments, the anti-Vγ4 antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 117-162. In some embodiments, the anti-Vγ4 antibody or fragment thereof may comprise a heavy-chain variable (VH) amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 117-139, preferably SEQ ID NO: 125. Alternatively, the anti-Vγ4 antibody or fragment thereof may comprise a light-chain variable (VL) amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 140-162, preferably SEQ ID NO: 148.

[0012] In some embodiments, the anti-Vγ4 antibody or a fragment thereof (a) A VH comprising HCDR1 having sequence number 79, HCDR2 having sequence number 56, and HCDR3 having sequence number 10, wherein the VH optionally comprises a VH having sequence number 125. A VL containing LCDR1 having sequence number 102, LCDR2 having sequence number A9 (in Figure 1), and LCDR3 having sequence number 33, wherein the VL optionally contains a VL with sequence number 148. (b) A VH comprising HCDR1 having sequence number 86, HCDR2 having sequence number 63, and HCDR3 having sequence number 17, wherein the VH optionally includes sequence number 132, A VL containing LCDR1 having sequence number 109, LCDR2 having sequence number A16 (in Figure 1), and LCDR3 having sequence number 40, wherein the VL optionally contains a VL with sequence number 155. (c) A VH comprising HCDR1 having sequence number 73, HCDR2 having sequence number 50, and HCDR3 having sequence number 4, wherein the VH may optionally be a VH comprising sequence number 119, A VL containing LCDR1 having sequence number 96, LCDR2 having sequence number A3 (Figure 1), and LCDR3 having sequence number 27, wherein the VL optionally contains a VL with sequence number 142. (d) A VH comprising HCDR1 having sequence number 83, HCDR2 having sequence number 60, and HCDR3 having sequence number 14, wherein the VH may optionally include a VH having sequence number 129. A VL comprising LCDR1 having sequence number 106, LCDR2 having sequence number A13 (in Figure 1), and LCDR3 having sequence number 37, wherein the VL optionally includes a VL containing sequence number 152. (e) A VH comprising HCDR1 having sequence number 84, HCDR2 having sequence number 61, and HCDR3 having sequence number 15, wherein the VH optionally comprises a VH having sequence number 130. A VL containing LCDR1 having sequence number 107, LCDR2 having sequence number A14 (Figure 1), and LCDR3 having sequence number 38, wherein the VL optionally contains a VL with sequence number 153. (f) A VH comprising HCDR1 having sequence number 88, HCDR2 having sequence number 65, and HCDR3 having sequence number 19, wherein the VH may optionally include a VH comprising sequence number 134. A VL containing LCDR1 having sequence number 111, LCDR2 having sequence number A18 (in Figure 1), and LCDR3 having sequence number 42, wherein the VL optionally contains a VL with sequence number 157. (g) A VH comprising HCDR1 having sequence number 92, HCDR2 having sequence number 69, and HCDR3 having sequence number 23, wherein the VH may optionally include a VH comprising sequence number 138. A VL containing LCDR1 having sequence number 115, LCDR2 having sequence number A22 (in Figure 1), and LCDR3 having sequence number 46, wherein the VL optionally contains a VL with sequence number 161. (h) A VH comprising HCDR1 having sequence number 71, HCDR2 having sequence number 48, and HCDR3 having sequence number 2, wherein the VH optionally comprises a VH having sequence number 117. A VL containing LCDR1 having sequence number 94, LCDR2 having sequence number A1 (Figure 1), and LCDR3 having sequence number 25, wherein the VL optionally contains a VL with sequence number 140. (i) A VH comprising HCDR1 having sequence number 72, HCDR2 having sequence number 49, and HCDR3 having sequence number 3, wherein the VH optionally comprises a VH having sequence number 118, A VL containing LCDR1 having sequence number 95, LCDR2 having sequence number A2 (in Figure 1), and LCDR3 having sequence number 26, wherein the VL optionally contains a VL with sequence number 141. (j) A VH comprising HCDR1 having sequence number 74, HCDR2 having sequence number 51, and HCDR3 having sequence number 5, wherein the VH optionally comprises a VH having sequence number 120. A VL containing LCDR1 having sequence number 97, LCDR2 having sequence number A4 (in Figure 1), and LCDR3 having sequence number 28, wherein the VL optionally contains a VL with sequence number 143. (k) A VH comprising HCDR1 having sequence number 75, HCDR2 having sequence number 52, and HCDR3 having sequence number 6, wherein the VH optionally comprises a VH having sequence number 121. A VL containing LCDR1 having sequence number 98, LCDR2 having sequence number A5 (Figure 1), and LCDR3 having sequence number 29, wherein the VL optionally contains a VL with sequence number 144. (l) A VH comprising HCDR1 having sequence number 76, HCDR2 having sequence number 53, and HCDR3 having sequence number 7, wherein the VH may optionally include a VH comprising sequence number 122. A VL containing LCDR1 having sequence number 99, LCDR2 having sequence number A6 (Figure 1), and LCDR3 having sequence number 30, wherein the VL optionally contains a VL with sequence number 145. (m) A VH comprising HCDR1 having sequence number 77, HCDR2 having sequence number 54, and HCDR3 having sequence number 8, wherein the VH may optionally be a VH comprising sequence number 123. A VL containing LCDR1 having sequence number 100, LCDR2 having sequence number A7 (in Figure 1), and LCDR3 having sequence number 31, wherein the VL optionally contains a VL with sequence number 146. (n) A VH comprising HCDR1 having sequence number 78, HCDR2 having sequence number 55, and HCDR3 having sequence number 9, wherein the VH optionally comprises a VH having sequence number 124. A VL containing LCDR1 having sequence number 101, LCDR2 having sequence number A8 (in Figure 1), and LCDR3 having sequence number 32, wherein the VL optionally contains a VL with sequence number 147. (o) A VH comprising HCDR1 having sequence number 80, HCDR2 having sequence number 57, and HCDR3 having sequence number 11, wherein the VH optionally has sequence number 126 Including VH, A VL containing LCDR1 having sequence number 103, LCDR2 having sequence number A10 (in Figure 1), and LCDR3 having sequence number 34, wherein the VL optionally contains a VL with sequence number 149. (p) A VH comprising HCDR1 having sequence number 81, HCDR2 having sequence number 58, and HCDR3 having sequence number 12, wherein the VH may optionally include a VH comprising sequence number 127. A VL containing LCDR1 having sequence number 104, LCDR2 having sequence number A11 (in Figure 1), and LCDR3 having sequence number 35, wherein the VL optionally contains a VL with sequence number 150. (q) A VH comprising HCDR1 having sequence number 82, HCDR2 having sequence number 59, and HCDR3 having sequence number 13, wherein the VH may optionally include a VH comprising sequence number 128. A VL containing LCDR1 having sequence number 105, LCDR2 having sequence number A12 (Figure 1), and LCDR3 having sequence number 36, wherein the VL optionally contains a VL with sequence number 151. (r) A VH comprising HCDR1 having sequence number 85, HCDR2 having sequence number 62, and HCDR3 having sequence number 16, wherein the VH may optionally include a VH comprising sequence number 131. A VL comprising LCDR1 having sequence number 108, LCDR2 having sequence number A15 (Figure 1), and LCDR3 having sequence number 39, wherein the VL optionally includes a VL containing sequence number 154. (s) A VH comprising HCDR1 having sequence number 87, HCDR2 having sequence number 64, and HCDR3 having sequence number 18, wherein the VH optionally comprises a VH having sequence number 133, A VL comprising LCDR1 having sequence number 110, LCDR2 having sequence number A17 (in Figure 1), and LCDR3 having sequence number 41, wherein the VL optionally includes a VL containing sequence number 156. (t) A VH comprising HCDR1 having sequence number 89, HCDR2 having sequence number 66, and HCDR3 having sequence number 20, wherein the VH may optionally include a VH comprising sequence number 135. A VL comprising LCDR1 having sequence number 112, LCDR2 having sequence number A19 (Figure 1), and LCDR3 having sequence number 43, wherein the VL optionally includes a VL containing sequence number 158. (u) A VH comprising HCDR1 having sequence number 90, HCDR2 having sequence number 67, and HCDR3 having sequence number 21, wherein the VH optionally comprises a VH having sequence number 136, A VL comprising LCDR1 having sequence number 113, LCDR2 having sequence number A20 (Figure 1), and LCDR3 having sequence number 44, wherein the VL optionally includes a VL containing sequence number 159. (v) A VH comprising HCDR1 having sequence number 91, HCDR2 having sequence number 68, and HCDR3 having sequence number 22, wherein the VH may optionally be a VH comprising sequence number 137, A VL containing LCDR1 having sequence number 114, LCDR2 having sequence number A21 (in Figure 1), and LCDR3 having sequence number 45, wherein the VL optionally contains a VL with sequence number 160. and / or, (w) A VH comprising HCDR1 having sequence number 93, HCDR2 having sequence number 70, and HCDR3 having sequence number 24, wherein the VH may optionally include a VH comprising sequence number 139. A VL containing LCDR1 having sequence number 116, LCDR2 having sequence number A23 (as shown in Figure 1), and LCDR3 having sequence number 47, wherein the VL optionally includes one or more VLs containing sequence number 162.

[0013] In some embodiments, the anti-Vγ4 antibody or a fragment thereof contains an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 163 to 185.

[0014] In some embodiments, the anti-Vγ4 antibody or a fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 233-255. In related embodiments, the anti-Vγ4 antibody comprises, or comprises, a heavy chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 284-306 and / or a light chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 307-329.

[0015] In some embodiments, an anti-Vγ4 antibody or fragment thereof specifically binds to the Vγ4 chain of the γδ T cell receptor (TCR) and competes for binding to the Vγ4 chain of the γδ T cell receptor (TCR) by the antibody or fragment thereof as defined herein.

[0016] A further aspect of the present invention provides a population of Vγ4 T cells obtained by an ex vivo method as defined herein.

[0017] A further aspect of the present invention provides a composition comprising a population of Vγ4 T cells as defined herein.

[0018] A further aspect of the present invention provides a pharmaceutical composition comprising a population of Vγ4 T cells as defined herein, together with an optionally pharmaceutically acceptable diluent or carrier.

[0019] A further aspect of the present invention provides a pharmaceutical composition of the present invention as defined herein for use as a pharmacopoeia. Similarly, a method is provided for treating a disease or disorder in a subject requiring such treatment (e.g., cancer, infectious disease or inflammatory disease), comprising administering a therapeutically effective amount of the anti-Vγ4 T cell population of the present invention or the pharmaceutical composition of the present invention as defined herein to the subject. [Brief explanation of the drawing]

[0020] [Figure 1] Exemplary complementarity-determining region (CDR) sequences of anti-Vγ4 antibodies. Exemplary CDR sequences of anti-Vγ4 antibodies described herein are shown. The corresponding sequence numbers are indicated to the right of each sequence. [Figure 2] Antibody specificity for heterodimeric TCR antigens by DELFIA Elisa assay. (A) Results for all antibodies that passed QC evaluation (SEC-HPLC for analysis) and also demonstrated specificity for human Vγ4 chains are shown. These antibodies (X axis) were tested for binding to four different recombinant heterodimeric human TCRs (DV1-GV4, DV2-GV4, DV1-GV2, DV1-GV8). Controls include isotype control anti-chicken lysozyme D1.3 antibody (proprietary, far left), as well as anti-Vδ1 antibodies REA173 (Miltenyi) and TS8.2 (Fisher) - far right. (B) Quantification of the data shown in (A), and further showing the increase in the multiplicity change of binding of each example clone to human Vγ4 chain versus human Vγ2 chain. [Figure 3]Comparison of antibodies binding to Vγ4Vδ1TCR, which is represented as either a recombinant antigen or a recombinant cell surface receptor. (A) Normalized and logarithmically transformed X / Y plots of antibodies binding to either DV1-GV4 antigen (Y axis) or JRT3-hu17 cells (X axis) via Delfia ELISA. The vertical gray dotted lines indicate the cutoffs for mAbs considered negative (left) and positive (right) for JRT3-hu17 binding in this experiment. The gMFI signal on the X axis was normalized to CD3 to account for the variation in TCR expression between each construct. (B) Flow data plots further showing the negative / positive cutoffs. Antibody G4_26 (center left figure) shows the highest normalized gMFI value in the negative group and shows a similar plot to the negative isotype control (D1.3, far left figure). G4_15 (center figure) has the lowest normalized gMFI value in the positive group and shows a weak but distinct enhancement of staining compared to the D1.3 isotype control. Examples of intermediate (G4_16, center right) and strong (G4_18, far right) signals are also provided for reference. [Figure 4] (A) Histogram representation of antibody binding to recombinant expressed γ4TCRs containing different CDR3 sequences and / or paired with different delta chains. Series analysis is shown below: Antibody binding signal to Vγ4Vδ1-hu17 (black bars), antibody binding signal to Vγ4Vδ1-hu20 (horizontal striped bars), antibody binding signal to Vγ4Vδ2hu20γ-PBδ (diagonal striped bars), antibody binding signal to Vγ4Vδ5-LES (white bars). All binding signals normalized to CD3 to explain the variation in TCR expression between different TCR constructs within JRT3 cells. (B) Example of flow data for the two lead antibodies in this experiment to further explain the difference between an exemplary antibody (G4_3) that showed positivity for all Vγ4TCRs and another lead antibody (G4_4) that showed positivity for only some Vγ4TCRs. [Figure 5]Antibody binding and epitope mapping to chimeric hu17 TCRs expressed in JRT3 cells. (A) Alignment of the germline-encoded gamma variable region of the shown chimeric hu17 constructs is presented. Due to space limitations, the first 10 amino acids of the mature Vγ2 / 3 / 4 sequence (amino acids 1-10 of SEQ ID NO: 256 [SSNLEGRTKS]) are omitted, but note that they are identical in all constructs. Different amino acids from the reference hu17 sequence (wild-type Vγ4 TCR) are shown. (B) Table summarizing the reactivity of each antibody to the shown chimeric TCR constructs. The results highlight the relative binding specificity of each antibody shown to individual TCRs expressed in JRT3 cells. (C) Example of epitope mapping flow data to explain the different binding signals observed in this experiment. [Figure 6] Examples of antibody binding and conferred function in Vγ4TCR(hu17) expressing cells. (A) Titration of antibodies to JRT3-hu17 showing all exemplary antibodies bound to JRT3-hu17 cells. Untransduced JRT3 cells (without TCR) used as a negative control, demonstrating that hu17 expression is essential for antibody binding. (B) Downregulation of TCR conferred by titration antibody versus positive control antibody: Downregulation conferred by anti-CD3ε (UCHT-1, Biolegend) or anti-pan-TCRγδ (B1, Biolegend). (C) Upregulation of CD69 conferred by titration antibody versus comparison antibody: Upregulation observed by anti-CD3ε (UCHT-1, Biolegend) or anti-pan-TCRγδ (B1, Biolegend). [Figure 7]Examples of antibody targeting and regulation of primary Vγ4-positive cells. (A) Titration of anti-Vγ4 antibodies to primary Vγ4+ T cells grown from the skin of two separate donors, demonstrating that all exemplary antibodies can bind to primary skin-derived Vγ4+ T cells in a dose-dependent manner. Isotype controls were used as negative controls to demonstrate the specificity of the exemplary antibodies to Vγ4. (B) Binding of anti-Vγ4 antibodies to peripheral blood mononuclear cell (PBMC)-derived Vγ4+ T cells, demonstrating that substantially all exemplary antibodies can bind to primary blood-derived Vγ4+ T cells. RSV isotype controls were used as negative controls. (C) Binding of anti-Vγ4 antibodies G4_3, G4_12, and G4_18 to intestinal intraepithelial lymphocytes (IELs) from colorectal cancer (CRC) patients, demonstrating that all three exemplary antibodies bind to this cell population. Cells were gated as single viable γδ+, IgG1+(Vγ4)+ cells. (D) Phenotype of Vγ4+γδT cells in intestinal digestate before stimulation with anti-Vγ4 antibody, demonstrating that Vγ4+ cells can be identified using the exemplary antibody G4_18. 1.4% of single viable cells were Vδ1+. Of these, 44.2% were paired with Vγ4, which displayed markers of tissue retention (CD69+CD103+). (E) Representative FACS plots showing TCR downcontrol conferred by exemplary antibodies G4_12 and G4_18, respectively, versus downcontrol conferred by isotype-negative controls. [Figure 8] Use of Vγ4-specific antibodies to increase the number of primary human Vγ4 T cells. (A) Exemplary flow data to illustrate the increase in Vγ4 T cells after 14 days of culture of plate-bound anti-Vγ4 clone G4_12 and PBMCs compared to isotype controls in the presence of IL-2 or IL-2+IL-15 (determined by staining with clone G4_18). (B) Summary of the increase in Vγ4 T cells after 7 days (top) and 14 days (bottom) of culture of plate-bound anti-Vγ4 clone G4_12 and PBMCs from two donors compared to isotype controls in the presence of IL-2 or IL-2+IL-15 (determined by staining with clone G4_18). [Modes for carrying out the invention]

[0021] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains. As used herein, the following terms have the meanings given below.

[0022] Gamma delta (γδ) T cells represent a subset of T cells that express distinct, typical T cell receptors (TCRs) on their surface. These TCRs consist of one gamma (γ) chain and one delta (δ) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region, and a cytoplasmic tail. The V region contains the antigen-binding site. Human γδ T cells have two main subtypes, one predominant in peripheral blood and the other in non-hematopoietic tissues. The two subtypes can be defined by the type of δ and / or γ present in the cell. For example, many γδ T cells in the blood express a Vδ2 TCR, e.g., Vγ9Vδ2, which is less common in γδ T cells in tissues; for example, skin more frequently uses Vδ1, and intestines more frequently uses Vγ4. "Vγ4 T cells" refer to γδ T cells with a Vγ4 chain, i.e., Vγ4+ cells.

[0023] "Gamma Variable 4" may also be referred to as Vγ4 or Vg4. A gamma variable 4 polypeptide, or a nucleotide encoding a TCR chain containing this region, or a TCR protein complex containing this region, may be referred to as "TRGV4". Any antibody or fragment that interacts with the Vγ4 chain of a γδTCR is, in effect, an antibody or fragment that binds to Vγ4 and may be referred to as "anti-TCR gamma variable 4 antibody or fragment" or "anti-Vγ4 antibody or fragment". A human Vγ4 polypeptide may mean a polypeptide having an amino acid sequence corresponding to amino acids 1-99 of SEQ ID NO: 1. This 99-amino acid sequence also corresponds to SEQ ID NO: 334. Therefore, it should be understood that references herein to amino acids 1-99 of SEQ ID NO: 1 may be used interchangeably with references to SEQ ID NO: 334 according to all aspects and embodiments of the present invention. For example, references herein to amino acid regions 67-82 of SEQ ID NO: 1 are equivalent to amino acid regions 67-82 of SEQ ID NO: 334 and may be used interchangeably herein.

[0024] "Delta Variable 1" may also be referred to as Vδ1 or Vd1. A delta variable 1 polypeptide, or a nucleotide encoding a TCR chain containing this region, or a TCR protein complex containing this region, may be referred to as "TRDV1". Any antibody or fragment that interacts with the Vδ1 chain of γδTCR is, in effect, an antibody or fragment that binds to Vδ1 and may be referred to as "anti-TCR delta variable 1 antibody or fragment" or "anti-Vδ1 antibody or fragment". Human Vδ1 polypeptide may mean a polypeptide having the amino acid sequence corresponding to SEQ ID NO: 337.

[0025] "Gamma Variable 2" may also be referred to as Vγ2 or Vg2. A gamma variable 2 polypeptide, or a nucleotide encoding a TCR chain containing this region, or a TCR protein complex containing this region, may be referred to as "TRGV2". Any antibody or fragment that interacts with the Vγ2 chain of the γδTCR is, in effect, an antibody or fragment that binds to Vγ2 and may be referred to as "anti-TCR gamma variable 2 antibody or fragment" or "anti-Vγ2 antibody or fragment". Human Vγ2 polypeptide may refer to a polypeptide having the amino acid sequence corresponding to SEQ ID NO: 335.

[0026] "Gamma Variable 8" may also be referred to as Vγ8 or Vg8. A gamma variable 8 polypeptide, or a nucleotide encoding a TCR chain containing this region, or a TCR protein complex containing this region, may be referred to as "TRGV8". Any antibody or fragment that interacts with the Vγ8 chain of the γδTCR is, in effect, an antibody or fragment that binds to Vγ8 and may be referred to as "anti-TCR gamma variable 8 antibody or fragment" or "anti-Vγ8 antibody or fragment". Human Vγ8 polypeptide may refer to a polypeptide having the amino acid sequence corresponding to SEQ ID NO: 336.

[0027] The term "antibody" includes any antibody protein construct that includes at least one antibody variable domain, which includes at least one antigen-binding site (ABS). Antibodies include, but are not limited to, IgA, IgG, IgE, IgD, and IgM type (and their subtypes) immunoglobulins. The overall structure of immunoglobulin G (IgG) antibodies assembled from two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides is well established and highly conserved in mammals (Padlan (1994) Mol.Immunol.31:169-217).

[0028] Conventional antibodies or immunoglobulins (Ig) are proteins containing four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy (H) chain variable domain is abbreviated herein as VH, and the light (L) chain variable domain is abbreviated herein as VL. These domains, their associated domains, and domains derived therefrom may be referred herein as immunoglobulin chain variable domains. The VH domains and VL domains (also referred to as VH regions and VL regions) may be further divided into regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Framework regions and complementarity-determining regions are clearly defined (Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition USD Department of Health and Human Services, (1991) NIH Publication Number 91-3242). There are also other numbering conventions for CDR sequences. For example, as described in Chothia et al. (1989) Nature 342:877-883. In conventional antibodies, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two deuteroglobulin chains and two light immunoglobulin chains are formed by deuteroglobulin and light immunoglobulin chains interconnected, for example, by disulfide bonds, and similarly connected heavy chains. The constant region of the heavy chain contains three domains CH1, CH2, and CH3. The constant region of the light chain consists of one domain CL. The variable domains of the heavy chain and the light chain are binding domains that interact with the antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0029] As used herein, an antibody fragment (which may also be referred to as an “antibody fragment,” “immunoglobulin fragment,” “antigen-binding fragment,” or “antigen-binding polypeptide”) is a portion of an antibody (or a construct containing such portion) that specifically binds to a target, or a gamma-variable 4 (Vγ4) chain of a γδ T cell receptor (for example, a molecule in which one or more immunoglobulin chains are not full length but specifically bind to a target). Examples of binding fragments included in the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain), (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments linked by disulfide bridges in the hinge region), (iii) Fd fragment (consisting of VH domain and CH1 domain), (iv) Fv fragment (consisting of a single group of VL and VH domains of the antibody), (v) Single-stranded variable fragment scFv (consisting of a VL domain and a VH domain linked by a synthetic linker, which enables the VL and VH domains to be paired and formed as a monovalent molecule using recombination), (vi) VH (variable immunoglobulin chain domain consisting of VH domains), (vii) VL (variable immunoglobulin chain domain consisting of VL domains), (viii) Domain antibody (consisting of either a dAb, VH domain, or VL domain), (ix) Minibody (consisting of a pair of scFv fragments joined via a CH3 domain), (x) Diabody (consisting of a non-covalent dimer of scFv fragments, each consisting of a VH domain from one antibody connected to a VL domain from another antibody by a small peptide linker).

[0030] "Human antibody" refers to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human subjects administered with the human antibody do not produce an interspecies antibody response (e.g., referred to as a HAMA-human anti-mouse antibody response) to the primary amino acids contained in the antibody. The human antibody may contain amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis or somatic mutation), for example, in the CDR, particularly CDR3. However, the term is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted into a human framework sequence. Human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transformed in a host cell, antibodies isolated from a recombinant combinatorial human antibody library, or antibodies isolated from an animal (e.g., a mouse) that has been genetically modified for the human immunoglobulin gene, or antibodies prepared, expressed, created, or isolated by any other means, including splicing of the human immunoglobulin gene sequence into another DNA sequence, may also be referred to as "recombinant human antibodies."

[0031] Replacing at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain with a corresponding residue from a human variable domain is called "humanization." Humanization of the variable domain can reduce human immunogenicity.

[0032] "Specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment of an antibody can bind. Antibody specificity is the antibody's ability to recognize a particular antigen as a unique molecular entity and distinguish it from other antigens. An antibody that "specifically binds" to an antigen or epitope is a well-understood term in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, more persistently and / or with higher affinity to a particular target antigen or epitope than it reacts with other targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, binding activity, and more readily and / or with higher persistence than it would to bind to other substances. If the binding is statistically significant compared to an unrelated binder, the antibody (or fragment thereof) may be considered to bind specifically to the target.

[0033] "Affinity," expressed by the equilibrium constant (KD) for the dissociation of an antigen and an antigen-binding polypeptide, is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antibody (or a fragment thereof). A smaller KD value indicates a stronger binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can be expressed as the affinity constant (KA), or 1 / KD. Affinity can be determined by known methods depending on the specific antigen of interest. For example, KD can be determined by surface plasmon resonance.

[0034] 10 -6 Any KD value less than 1 is considered to indicate binding. Specific binding of an antibody or fragment to an antigen or antigenic determinant can be determined by any suitable known method, including, for example, scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays), as well as various variations of these known in the Art.

[0035] "Binding activity" is a measure of the strength of binding between an antibody or fragment of an antibody and its associated antigen. Binding activity is related to both the affinity between the antigenic determinant and its antigen-binding site on the antibody, and the number of suitable binding sites present on the antibody.

[0036] "Human tissue Vγ4+ cells," "hematopoietic and blood Vγ4+ cells," and "tumor-infiltrating lymphocytes (TILs) Vγ4+ cells" are defined as Vγ4+ cells contained in or derived from human tissue, hematopoietic blood system, or human tumor, respectively. All of the aforementioned cell types can be identified by (i) their location or the place from which they originate, and (ii) their expression of Vγ4+ TCRs.

[0037] Appropriately, an antibody or a fragment thereof (i.e., a polypeptide) is isolated. An “isolated” polypeptide is one that has been removed from its original environment. The term “isolated” can also be used to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody or fragment that specifically binds to Vγ4 substantially does not contain antibodies that specifically bind to other antigens other than Vγ4). The term “isolated” can also be used to refer to a preparation in which, when formulated as an active ingredient in a pharmaceutical composition, the isolated antibody is pure enough to be therapeutically administered, or at least 70%–80% (w / w), more preferably at least 80%–90% (w / w), even more preferably 90–95%, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w).

[0038] Ideally, the polynucleotides used in this invention are isolated. “Isolated” polynucleotides are those removed from their original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the materials that coexist in the natural system. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment, or if it is contained within cDNA.

[0039] Antibodies or fragments thereof may be “functionally active variants,” including naturally occurring allele variants and mutants or any other non-naturally occurring variants. As is known in the art, allele variants essentially alter the biological function of polypeptides. This is an alternative form of (poly)peptide characterized by having one or more amino acid substitutions, deletions, or additions. As a non-limiting example, the functionally active variant may still function when the framework containing the CDR is modified, when the CDR itself is modified, when the CDR is grafted onto an alternative framework, or when an N-terminal or C-terminal extension is incorporated. Furthermore, the CDR-containing binding domain may be paired with a different partner chain, such as one shared with another antibody. When shared with a so-called “common” light chain or “common” heavy chain, the binding domain may still function. Furthermore, the binding domain may function when polymerized. Furthermore, “antibodies or fragments thereof” may also include functional variants in which the VH, VL, or constant domain is modified away from or toward a different canonical sequence (e.g., as described on IMGT.org) and still functions.

[0040] For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between the first polypeptide sequence and the second polypeptide sequence can be calculated using NCBI BLASTv2.0 with a standard polypeptide sequence setting (BLASTP). For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between the first nucleotide sequence and the second nucleotide sequence can be calculated using NCBI BLASTv2.0 with a standard nucleotide sequence setting (BLASTN).

[0041] Polypeptide sequences or polynucleotide sequences are said to be the same as, or "identical to," other polypeptide or polynucleotide sequences if they share 100% sequence identity throughout their entire length. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of a polypeptide, and from the 5' end to the 3' end of a polynucleotide.

[0042] In some embodiments, sequence identity of any particular percentage of the sequence is calculated without the sequences of all six CDRs of the antibody. For example, an anti-Vγ4 antibody or its antigen-binding fragment may contain a variable heavy chain region sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular variable heavy chain region sequence, and / or a variable light chain region sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular variable light chain region sequence, and any amino acid mutations occur only in the framework regions of the variable heavy chain region sequence and the variable light chain region sequence. In such embodiments, an anti-Vγ4 antibody or fragment having a specific sequence identity retains the complete heavy and light chain CDR1, CDR2, and CDR3 sequences of the corresponding anti-Vγ4 antibody or fragment.

[0043] A “difference” between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at the position of the second sequence compared to the first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. An insertion, deletion, or substitution in the second sequence, which is otherwise identical to the first sequence (100% sequence identity), reduces the percentage of sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in 88.9% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share more than 66% identity (they share 66.7% identity).

[0044] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. "Addition" refers to the addition of a single amino acid residue to the sequence of the first polypeptide. "Addition" is the addition of a group (including addition at either end of the first polypeptide). "Substitution" is the replacement of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. The substitution may be conserved or non-conserved. "Deletion" is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletion at either end of the first polypeptide).

[0045] Using three-letter and one-letter notations, naturally occurring amino acids are represented as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine ​​(C or Cys), methionine (M or Met), serine (S or Ser), and threonine (T or Thr). If a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. If the residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. Unless otherwise specified in the context, references to aspartic acid include aspartate, and references to glutamic acid include glutamate.

[0046] A “conservative” amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and is expected to have little effect on the function, activity, or other biological properties of the polypeptide. Such a conservative substitution is, appropriately speaking, one in the following group of amino acids that is replaced by another amino acid residue from the same group:

[0047] [Table 1]

[0048] Preferably, the hydrophobic amino acid residue is a nonpolar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C. In some embodiments, the hydrophobic amino acid residue is selected from glycine, alanine, valine, methionine, leucine, isoleucine, phenylalanine, tyrosine, or tryptophan.

[0049] For use herein, polypeptide sequence numbering and the definitions of CDR and FR are as defined according to the Kabat system (Kabat et al., 1991, which is incorporated herein by reference in its entirety). The "corresponding" amino acid residues between a first polypeptide sequence and a second polypeptide sequence are those amino acid residues in the first sequence that share the same position as the amino acid residues in the second sequence according to the Kabat system, whereas the second sequence may have different identity from the first sequence. If the framework and CDR are of the same length according to the Kabat definition, appropriately corresponding residues share the same number (and letter). Alignment can be achieved manually or by using known computer algorithms for sequence alignment, such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0050] In this specification, “epitope” refers to a portion of a target to which an antibody or a fragment of it specifically binds. An epitope may also be called an “antigenic determinant.” When two antibodies recognize the same or sterically overlapping epitopes, they bind to “essentially the same epitope” as another antibody. A method commonly used to determine whether two antibodies bind to the same or overlapping epitopes is a competitive assay. This involves using labeled antigens or labeled antibodies. Antibodies can be composed of multiple different forms using any of the body (e.g., well plates with radiolabeling or enzymatic labeling, or flow cytometry in antigen-expressing cells). Antibodies bind to the "same epitope" as another antibody if both recognize the same epitope (i.e., all contact points between the antigen and antibody are the same). For example, if all contact points in a particular region of the antigen are identified as the same using characterization methods such as antibody / antigen crosslinking MS, HDX, X-ray crystallography, cryo-electron microscopy, or mutagenesis, then an antibody may bind to the same epitope as another antibody.

[0051] Furthermore, by utilizing such characterization methods, it is also possible to characterize antibodies that bind to essentially the same epitopes by recognizing some, but not all, of the identical contact points. Specifically, such antibodies may share a sufficient number of identical contact points in a particular antigenic region, providing nearly equivalent technical effects and / or equivalent selectivity for antigen interaction. Moreover, in some cases where antibodies recognize essentially the same epitopes and confer nearly equivalent technical effects and / or selectivity for interaction, it may also be useful to define the epitope binding footprint by the entire antigen contact, including from the N-terminal antigen contact point to the C-terminal antigen contact point.

[0052] Epitopes found in protein targets can be defined as "linear epitopes" or "contaxial epitopes." Linear epitopes are formed by a continuous sequence of amino acids in a protein antigen. Contaxial epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0053] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial replication origins, as well as episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can mediated the expression of the gene to which they are operably bound. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vector. However, it is intended to include viral vectors that perform equivalent functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), as well as other forms of expression vectors such as bacteriophages and phagemid systems. As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to the cell into which the recombinant expression vector has been introduced. Such terminology is intended to refer not only to specific target cells, but also, for example, to the offspring of such cells if the offspring are subsequently used to create a cell line or cell bank that may be optionally stored, provided, sold, transferred, or used to produce antibodies or fragments thereof as described herein.

[0054] "Subject," "patient," or "individual" refers to the subject being treated, particularly a mammalian subject. Mammalian subjects include humans, non-human primates, livestock (such as cattle), sports animals, or companion animals such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is human. In alternative embodiments, the subject is a non-human mammal such as a mouse.

[0055] The term "sufficient amount" means an amount sufficient to produce the desired effect. The term "therapeutic amount" is an amount that is effective in improving the symptoms of a disease or disorder. Since prevention can be considered a treatment, a therapeutic amount may also be a "preventive amount."

[0056] A disease or disorder is considered “improved” if the severity of the signs or symptoms of the disease or disorder, the frequency with which such signs or symptoms are experienced by the subject, or both, decreases.

[0057] As used herein, “treating a disease or disorder” means reducing the frequency and / or severity of at least one sign or symptom of the disease or disorder experienced by the subject.

[0058] As used herein, “cancer” refers to the abnormal proliferation or division of cells. Generally, the proliferation and / or lifespan of cancer cells is greater than and uncoordinated with the proliferation and / or lifespan of the surrounding normal cells and tissues. Cancer can be benign, premalignant, or malignant. Cancer can occur in a variety of cells and tissues.

[0059] "Inflammation" refers to the chronic or acute induction of the immune system that results in inflamed cells, cell types, tissues, or organs.

[0060] As used herein, the term “approximately” includes values ​​that are 10% greater than and 10% less than the specified value, preferably including values ​​that are 5% greater than and 5% less than the specified value, and especially including the specified value. The term “between” includes the specified boundary values.

[0061] Methods for regulating γδT cells According to a first aspect of the present invention, an ex vivo method for modulating gamma variable 4-chain (Vγ4) T cells is provided, comprising administering an anti-Vγ4 antibody or a fragment thereof, as defined herein, to a cell population comprising Vγ4 T cells. It will be understood that "administering" the antibody or a fragment thereof includes "contacting" the Vγ4 T cells.

[0062] For the regulation of Vγ4T cells, - For example, the proliferation of Vγ4T cells by selectively increasing the number of Vγ4T cells or promoting the survival of Vγ4T cells. - For example, stimulating Vγ4T cells by increasing Vγ4T cell capacity, i.e., increasing target cell killing, - For example, preventing Vγ4T cell depletion by increasing the persistence of Vγ4T cells. -Degranulation of Vγ4T cells, -For example, immunosuppression of Vγ4 T cells by downregulating Vγ4 TCR cell surface expression, i.e., by causing internalization of Vγ4 TCR or a decrease in Vγ4 TCR protein expression, or by blocking Vγ4 TCR from binding. -This may include, for example, a reduction in the number of Vγ4T cells by suppressing Vγ4T cell proliferation or by inducing Vγ4T cell death (i.e., killing Vγ4T cells).

[0063] Such regulation of Vγ4 T cells may include, for example, activation or inhibition of Vγ4 T cells. In one embodiment, Vγ4 T cells are activated by administering an anti-Vγ4 antibody or a fragment thereof as defined herein. In an alternative embodiment, Vγ4 T cells are activated by administering an anti-Vγ4 antibody or a fragment thereof as defined herein. They are suppressed by [the specified agent]. In an alternative embodiment, Vγ4 T cells are not suppressed upon administration of an anti-Vγ4 antibody or fragment thereof as defined herein.

[0064] In one embodiment, the regulation of Vγ4 T cells involves administering an anti-TCR gamma 4 variable antibody or a fragment thereof to Vγ4 T cells in culture (i.e., in vitro or ex vivo). Vγ4 T cells may be present in a mixed cell population, for example, a cell population containing other lymphocyte cell types (e.g., αβ T cells or NK cells).

[0065] In one embodiment, a cell population containing Vγ4 T cells is isolated (i.e., from a sample described herein) before administration of an anti-Vγ4 antibody or a fragment thereof. In a further embodiment, the cell population is enriched with T cells before administration of an anti-Vγ4 antibody or a fragment thereof. In a further embodiment, the cell population is enriched with γδ T cells before administration of an anti-Vγ4 antibody or a fragment thereof.

[0066] The method may also be carried out with a cell population containing a purified fraction of γδT cells. In such embodiments, the cell population is depleted of non-γδT cell cell types, such as αβT cells and / or NK cells, present in the sample prior to administration of the anti-Vγ4 antibody or a fragment thereof. The cell population may also be rich in Vγ4-containing cell types, such as T cells and / or γδ cells, prior to administration of the anti-Vγ4 antibody or a fragment thereof. For example, before culturing the sample, the sample can be enriched with T cells, or enriched with γδT cells, or depleted with αβT cells, or depleted with non-γδT cells. In one embodiment, the sample is first depleted with αβT cells and then enriched with CD3+ cells. Enrichment or depletion can be achieved using techniques known in the art, such as using magnetic beads coated with antibodies that bind to molecules on the cell surface related to the phenotype to be enriched / depleted.

[0067] The presence of cell types other than lymphocytes in cell culture can inhibit the proliferation of Vγ4 cells. Such cells, such as stromal, epithelial, tumor, and / or supporting cells, can be removed before culture. Therefore, in one embodiment, the cell population does not come into direct contact with stromal cells during culture. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematological tumor cells. Preferably, lymphocytes do not come into direct contact with fibroblasts during culture. In one embodiment, the cell population does not come into direct contact with epithelial cells during culture. In one embodiment, the cell population does not come into direct contact with tumor cells and / or supporting cells during culture.

[0068] In one embodiment, the method comprises culturing Vγ4T cells in the absence of substantial contact with stromal cells. In a further embodiment, the method comprises culturing Vγ4T cells in the absence of substantial contact with fibroblasts.

[0069] In one embodiment, the method comprises culturing Vγ4T cells in a culture medium substantially free of serum (e.g., serum-free medium or medium containing a serum substitute (SR)). Thus, in one embodiment, the method comprises culturing in serum-free medium. Such serum-free medium may also include serum substitute medium, where the serum substitute is based on chemically defined components to avoid the use of human or animal-derived serum. In an alternative embodiment, the method comprises culturing in a culture medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the medium contains a serum substitute. In one embodiment, the medium does not contain any animal-derived products.

[0070] It will be understood that samples cultured in serum-free media have the advantage of avoiding problems related to filtration, precipitation, contamination, and serum supply. Furthermore, animal-derived products are clinically gray. It is not suitable for use in the manufacture of human therapeutic drugs.

[0071] In one embodiment, the anti-Vγ4 antibody or fragment thereof is in a soluble or immobilized form. For example, the antibody or fragment thereof may be administered to Vγ4 T cells in a soluble form. Alternatively, if the antibody or fragment thereof is bound or covalently bound to a surface such as beads or a plate (i.e., in an immobilized form), the antibody or fragment thereof may be administered to Vγ4 T cells. In one embodiment, the antibody is immobilized on a surface such as an Fc-coated well. Alternatively, the antibody or fragment thereof is bound to the surface of a cell (e.g., immobilized on the surface of an antigen-presenting cell (APC)). In another embodiment, the antibody is not immobilized on a surface when a population of cells comes into contact with it.

[0072] Cell populations that come into contact with the anti-Vγ4 antibody or a fragment thereof can be obtained from various types of samples (isolation methods are further described below). In one embodiment, the sample is a non-hematopoietic tissue sample. References to “non-hematopoietic tissue” or “non-hematopoietic tissue sample” herein include skin (e.g., human skin) and intestines (e.g., human intestines). Non-hematopoietic tissue is tissue other than blood, bone marrow, lymphoid tissue, lymph node tissue, or thymic tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In some embodiments, the cell population (e.g., γδT cells) is not obtained from a sample of a specific type of bodily fluid, such as blood or synovial fluid. In some embodiments, the cell population (e.g., γδT cells) is obtained from skin (e.g., human skin), which can be obtained by methods known in the art. For example, the cell population can be obtained from a non-hematopoietic tissue sample by culturing the non-hematopoietic tissue sample on a synthetic scaffold configured to facilitate the release of cells from the non-hematopoietic tissue sample. Alternatively, the method can be applied to cell populations (e.g., γδT cells) obtained from the gastrointestinal tract (e.g., colon or intestine), mammary glands, lungs, prostate, liver, spleen, pancreas, uterus, vagina, and other skin, mucous membranes, or serosal membranes.

[0073] In alternative embodiments, the sample is a hematopoietic sample or a fraction thereof (i.e., the cell population is obtained from the hematopoietic sample or a fraction thereof). References herein to “hematopoietic sample” or “hematopoietic tissue sample” include blood (such as peripheral blood or umbilical cord blood), bone marrow, lymphoid tissue, lymph node tissue, thymic tissue, and fractions or concentrated portions thereof. The sample is preferably blood containing peripheral blood or umbilical cord blood or fractions thereof, including buffy coat cells, leukocyte-deficient products, peripheral blood mononuclear cells (PBMCs) and low-density mononuclear cells (LDMCs). In some embodiments, the sample is human blood or a fraction thereof. Cells can be obtained from a blood sample using techniques known in the art, such as density gradient centrifugation. For example, whole blood is layered on equal volumes of FICOLL-HYPAQUE and subsequently centrifuged at 400xg for 15-30 minutes at room temperature. The interfacial material contains low-density mononuclear cells that can be collected and washed with culture medium and centrifuged at 200xg for 10 minutes at room temperature.

[0074] Cell populations can be obtained from cancer tissue samples, such as tumors of the intestines, breasts, or prostates (i.e., γδT cells may also be present in cancer tissue samples). In some embodiments, cell populations may originate from human cancer tissue samples (e.g., solid tumor tissue). In other embodiments, cell populations may originate from samples other than human cancer tissue (e.g., tissue that does not contain a substantial number of tumor cells). For example, cell populations may originate from areas of skin (e.g., healthy skin) that are distant from nearby or adjacent cancer tissue. Therefore, in some embodiments, cell populations are not obtained from cancer tissue (e.g., human cancer tissue).

[0075] Cell populations can be obtained from human or non-human animal tissue. Therefore, the method may further include the step of obtaining cell populations from human or non-human animal tissue. In one embodiment, the sample is obtained from a human. In an alternative embodiment, the sample is obtained from a non-human animal subject.

[0076] Proliferation of γδT cells In one embodiment, regulation includes activation of Vγ4T cells, particularly proliferation of Vγ4T cells. Accordingly, according to one aspect of the present invention, an ex vivo method for proliferation of Vγ4T cells is provided, comprising administering an anti-Vγ4 antibody or a fragment thereof, as defined herein, to a cell population comprising Vγ4T cells. Such proliferation of Vγ4T cells can be achieved by a selective increase in the number of Vγ4T cells and / or by promoting the survival of Vγ4T cells. In one embodiment, proliferation of Vγ4T cells comprises administering an anti-TCR gamma 4 variable antibody or a fragment thereof to Vγ4T cells in culture (i.e., in vitro or ex vivo). Vγ4T cells may be present in a mixed cell population, for example, a cell population comprising other lymphocyte cell types (e.g., αβT cells or NK cells).

[0077] Accordingly, the present invention provides an ex vivo method for producing an enriched population of γδT cells (e.g., Vγ4T cells). The enriched population can be produced from a mixed cell population isolated by a method comprising contacting the mixed cell population or a purified fraction thereof with an antibody or a fragment thereof (e.g., obtained from a sample taken from a parent / donor). The antibody (or fragment thereof) selectively promotes the proliferation of Vγ4T cells by binding to an epitope specific to the Vγ4 chain of the γδTCR.

[0078] Furthermore, a proliferated Vγ4 T cell population obtained according to a method defined herein is also provided. According to this aspect of the present invention, it will be understood that such a proliferated population of Vγ4 T cells may be obtained and / or proliferated in vitro or ex vivo. In one embodiment, a proliferated Vγ4 population obtained according to a method defined herein is provided, the Vγ4 population may be isolated and proliferated in vitro or ex vivo.

[0079] The antibodies or fragments thereof described herein may be used in methods for growing γδT cells (e.g., Vγ4T cells). These methods can be carried out in vitro. When the growth method is carried out in vitro, the antibodies (or fragments thereof) may be applied to isolated γδT cells (e.g., Vγ4T cells) obtained as described above. In some embodiments, γδT cells are grown from a population of cells isolated from a non-hematopoietic tissue sample. In alternative embodiments, γδT cells are grown from a population of cells isolated from a hematopoietic tissue sample, such as a blood sample.

[0080] Proliferation of γδT cells (e.g., Vγ4T cells) may involve culturing a sample in the presence of an antibody or fragment thereof as described herein, and cytokines. Cytokines may include interleukins, lymphokines, interferons, colony-stimulating factors, and chemokines. In one embodiment, the cytokines are selected from the group consisting of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-33 (IL-33), insulin-like growth factor 1 (IGF-1), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and stromal cell-derived factor-1 (SDF-1). References to cytokines in this specification may include, but are not limited to, any compounds having the same activity as the cytokines in terms of their ability to promote similar physiological effects on Vγ4T cells in culture, including mimics or their functional equivalents.

[0081] In one embodiment, the cytokine is a common cytokine receptor gamma chain (γc) family of cytokines. In a further embodiment, the γc cytokine is IL Selected from IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-21, or a mixture thereof.

[0082] The cytokine used (e.g., interleukin) may be of human or animal origin, and preferably of human origin. It may be a wild-type protein, or any biologically active fragment or variant, i.e., one that can bind to its receptor. Such binding may induce activation of γδT cells under the conditions of the method according to the present invention. More preferably, the cytokine may be in a soluble form fused or complexed with another molecule, such as a peptide, polypeptide, or biologically active protein. Preferably, human recombinant cytokines are used. More preferably, the interleukin concentration may vary between 1 and 10,000 U / ml, and even more preferably between 100 and 1,000 U / ml.

[0083] In further embodiments, the cytokine is a chemokine. It will be further understood that the chemokine varies and is selected depending on the sample used to obtain γδT cells.

[0084] In one embodiment, the method comprises culturing a cell population in the presence of IL-2, IL-9, and / or IL-15. In a further embodiment, the method comprises culturing a cell population in the presence of IL-2. In one embodiment, the method comprises culturing a cell population in the presence of IL-2 and / or IL-15 (i.e., IL2, IL-15, or a combination thereof). In an alternative embodiment, the method comprises culturing a cell population in the presence of IL-9 and / or IL-15 (i.e., IL9, IL-15, or a combination thereof). In one embodiment, the method comprises a cell population in the presence of IL-2, IL-9, and / or IL-15, as well as an additional growth factor (e.g., IL-21). In another embodiment, the method comprises culturing a cell population in a medium lacking growth factors other than IL-2 and / or IL-15. In an alternative embodiment, the method comprises culturing a cell population in a medium lacking growth factors other than IL-9 and / or IL-15. In a further embodiment, the method comprises culturing a cell population in a medium consisting of a basic medium supplemented with IL-2, IL-9, and / or IL-15.

[0085] In one embodiment, the method involves culturing a cell population in the presence of IL-15 and a factor selected from the group consisting of IL-2, IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, IFN-γ, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1).

[0086] The proliferation of γδ T cells may involve culturing a sample in the presence of at least one additional T cell mitogen. The term “T cell mitogen” (also called “γδ TCR agonist”) means any agent capable of stimulating T cells via TCR signaling, including but not limited to plant lectins such as phytohemagglutinin (PHA) and concanavalin A (ConA), as well as non-plant-derived lectins. In one embodiment, the T cell mitogen is an anti-CD3 monoclonal antibody (mAb). Other mitogens include phorbol 12-myristate-13-acetate (TPA) and its related compounds such as mezerein, or bacterial compounds (e.g., Staphylococcus enterotoxin A (SEA) and Streptococcus protein A). The T cell mitogen may be soluble or immobilized, and more than one T cell mitogen may be used in the proliferation method.

[0087] As used herein, the references to “proliferated” or “proliferated γδT cell population” include cell populations that are larger than or contain a greater number of cells than a non-proliferated population. Such populations may be majority, minority, or mixed populations with proliferation of a proportion or particular cell type within the population. The term “proliferation method” should be understood to refer to the process that results in a proliferated or proliferated population. Thus, a proliferated or proliferated population may contain a greater number or more cells than a population that has not undergone a proliferation process or a population prior to a proliferation process. Any numbers used herein to indicate proliferation (e.g., duplicating or polyploidy) indicate the number or size of a cell population or an increase in the number of cells, and represent the amount of proliferation.

[0088] In one embodiment, the method comprises culturing a cell population for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, 14 to 21 days, or about 14 days). In a further embodiment, the method comprises culturing a cell population for at least 7 days, e.g., at least 11 days or at least 14 days.

[0089] In a further embodiment, the method comprises culturing a cell population for a certain period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, 14 to 21 days, or about 14 days) in an amount effective to produce a proliferated γδT cell population.

[0090] In one embodiment, the cell population is cultured for a period of 5 to 60 days, such as 7 to 45 days, 7 to 21 days, 7 to 18 days, or 7 to 14 days. If the method includes an isolation culture period (e.g., 1 to 40 days, such as 14 to 21 days), the isolation and proliferation steps may last for 21 to 39 days in some embodiments.

[0091] The method may include the periodic addition of anti-Vγ4 antibody or fragments thereof and / or growth factors during culture. For example, anti-Vγ4 antibody or fragments thereof and / or growth factors may be added every 2 to 5 days, more preferably every 3 to 4 days. In one embodiment, anti-Vγ4 antibody or fragments thereof and / or growth factors are added after 7 days of culture, and thereafter every 2 to 3 days.

[0092] The proliferation method provides a population of proliferated γδT cells that is larger in number than the reference population. In some embodiments, the proliferated γδT cell population (e.g., Vγ4T cells) is larger in number than the isolated population of γδT cells before the proliferation process (e.g., at least twice, at least five times, at least ten times, at least 25 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 1,000 times or more compared to the isolated population of γδT cells before the proliferation process). In one embodiment, the proliferated γδT cell population (e.g., Vγ4T cells) is larger in number than the population cultured for the same time without the presence of an antibody or fragment thereof. In some embodiments, the proliferated γδT cell population (e.g., Vγ4T cells) is larger in number than the population cultured for the same time without the presence of an antibody or fragment thereof. The number of organisms will be higher than in a population cultured for the same amount of time in the presence of an isotype control.

[0093] The proliferation method provides a proliferated Vγ4T cell population having a higher proportion of Vγ4T cells than the reference population. In some embodiments, the proliferated Vγ4T cell population contains more than about 50% Vγ4T cells, for example, more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, or 95% Vγ4T cells. In further embodiments, the proliferated Vγ4T cell population contains more than about 60% Vγ4T cells, such as more than about 70% Vγ4T cells.

[0094] Numerous basal media suitable for use in the proliferation of γδ T cells are available, particularly AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies), EXVIVO-10, EXVIVO-15, or EXVIVO-20 (Lonza) in the presence of serum or plasma. The media may be supplemented with other media factors as defined herein, such as serum, serum proteins, and selective agents such as antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM, Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser, 1XMEM non-essential amino acids (Life Technologies)) and / or 10 μl / L β-mercaptoethanol. In some embodiments, the medium comprises RPMI-1640 supplemented with 5% human AB serum, sodium pyruvate (1 mM, Life Technologies), and penicillin / streptomycin. In alternative embodiments, AIM-V medium may be supplemented with CTS immunoserum substitute and amphotericin B. In certain embodiments, the medium may be further supplemented with IL-2, IL-4, IL-9, and / or IL-15 as described herein. Conveniently, during isolation and / or growth, the cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 in a suitable medium.

[0095] The addition of other factors to the proliferation culture of γδT cells may also be used. In one embodiment, such factors are used in proliferation to selectively promote the proliferation of γδT cells. For example, proliferation may further include the addition of exogenous cytokines such as interleukins to the proliferation culture. Such proliferation may include culturing γδT cells in the presence of IL-2 and IL-15. Alternatively, proliferation may include culturing γδT cells in the presence of IL-9 and IL-15. It will be understood that any proliferation step is carried out for a period of time effective in producing a proliferated population of γδT cells.

[0096] Methods for proliferating γδT cells may include population doubling times of less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0097] Method for isolating γδT cells As described herein, an antibody (or fragment thereof) may be applied to γδT cells in culture, i.e., γδT cells obtained from a sample. In one embodiment, a cell population is isolated from a sample before administration of the anti-Vγ4 antibody or fragment thereof. Thus, a method is provided for modulating (in particular, proliferating) Vγ4T cells, comprising administering the anti-Vγ4 antibody or fragment thereof, as defined herein, to a population of γδT cells isolated from a sample (e.g., a cell population containing Vγ4T cells).

[0098] In this specification, references to “isolation” or “isolating” cells, particularly γδT cells, refer to a method or process by which cells are removed, separated, purified, concentrated, or otherwise extracted from a tissue or pool of cells. It should be understood that such references include terms such as “isolated,” “removed,” “purified,” and “concentrated.” Isolation of γδT cells includes isolation or separation of cells from intact non-hematopoietic tissue samples or from stromal cells of non-hematopoietic tissue (e.g., fibroblasts or epithelial cells). Such isolation may, or in addition, include isolation or separation of γδT cells from other hematopoietic cells (e.g., αβT cells or other lymphocytes). Isolation may be a defined period, beginning, for example, when the tissue explant or biopsy is placed in an isolation culture and ending, for example, when the cells are collected from the culture by centrifugation or other means for transferring the isolated cell population to a growth culture, or when the cells are used for other purposes, or when the original tissue explant or biopsy is removed from the culture. The isolation process may last at least about 3 days to about 45 days. In one embodiment, the isolation process is at least about 10 to at least 28 days. In a further embodiment, the isolation process is at least 14 to at least 21 days. Thus, the isolation process may be at least 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, about 35, about 40, or about 45 days. It can be understood that cell proliferation may not be substantial in this isolation process, but it is not necessarily absent. Indeed, those skilled in the art will recognize that isolated cells may also begin to divide, producing multiple such cells in the isolation vessel containing the sample.

[0099] Therefore, references herein to “isolated γδT cells,” “isolated γδT cell population,” or “isolated population of γδT cells” should be understood to refer to γδ cells isolated, separated, removed, purified, or concentrated from a sample such as the original non-hematopoietic tissue sample, so as not to come into substantial contact with the cells contained within the intact (non-hematopoietic tissue) sample. References herein to “isolated Vγ4T cells,” “isolated Vγ4T cell population,” “isolated population of Vγ4T cells,” “removed Vγ4T cells,” “removed Vγ4T cell population,” or “removed population of Vγ4T cells” should be understood to refer to Vγ4T cells isolated, separated, removed, purified, or concentrated from a sample such as the original non-hematopoietic tissue sample, so as not to come into substantial contact with the cells contained within the intact (non-hematopoietic tissue) sample.

[0100] Non-hematopoietic tissue lymphocytes can be collected and isolated from stromal cells, such as cutaneous fibroblasts, by, for example, reliably pipetting them. The collected lymphocytes may be further washed through a 40 μm nylon mesh to retain any fibroblast aggregates that may have loosened during processing. Lymphocytes can also be isolated using, for example, fluorescence or magnetic-associated cell sorting with CD45 antibodies.

[0101] Isolation of γδT cells may involve culturing a sample in the presence of at least one cytokine. For example, the method may involve culturing a sample in the presence of at least one drug, such as a chemokine. It will be further understood that the chemokine is selected depending on the γδT cells to be isolated. Furthermore, the chemokine will vary and be selected depending on the sample used for the isolation of γδT cells.

[0102] Isolation of γδT cells may involve further culturing the sample in the presence of at least one cytokine, which may be different from the cytokine used in the initial culture.

[0103] The isolation method may include culturing the sample. References to “cultivate” herein include adding a sample, containing cells isolated, separated, removed, purified or concentrated from the sample, to a culture medium containing growth factors and / or essential nutrients required and / or preferred by the cells and / or sample. It will be understood that such culture conditions may be adapted according to the cells or cell populations isolated from the sample, or according to the cells or cell populations isolated and grown from the sample.

[0104] In certain embodiments, the culture period of the sample is sufficient to isolate γδT cells from the sample. In certain embodiments, the culture period is at least 14 days. In certain embodiments, the culture period is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culture period is between 14 and 35 days, such as between 14 and 21 days. In yet another further embodiment, the culture period is approximately 21 days.

[0105] In certain embodiments, γδT cells are collected from the culture after culturing the sample. Collection of γδT cells may include physical collection of γδT cells from the culture, isolation of γδT cells from other lymphocytes (e.g., αβT cells and / or NK cells), or isolation and / or separation of γδT cells from other cells present in the sample, such as stromal cells including fibroblasts. In one embodiment, γδT cells are collected by mechanical means (e.g., by pipetting). In a further embodiment, γδT cells are collected by magnetic separation and / or labeling. In yet another further embodiment, γδT cells are collected by flow cytometry techniques such as FACS. Thus, in certain embodiments, γδT cells are collected by specifically labeling the γδT cells. It will be understood that such collection of γδT cells may include physical removal of the sample from the culture, transfer to another culture vessel, or transfer to different or different culture conditions.

[0106] It will be understood that such collection of γδT cells is performed after a sufficient amount of time has elapsed to obtain an isolated population of γδT cells from the sample. In certain embodiments, γδT cells are collected at least one week, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after culturing the sample. Preferably, γδT cells are collected after 40 days or less, such as 38 days or less, 36 days or less, 34 days or less, 32 days or less, 30 days or less, 28 days or less, 26 days or less, or 24 days or less. In one embodiment, γδT cells are collected at least 14 days after culturing the sample. In further embodiments, γδT cells are collected 14 to 21 days after culturing the sample.

[0107] In one embodiment, the sample is cultured in a substantially serum-free medium (e.g., a serum-free medium or a medium containing a serum substitute (SR)). Thus, in one embodiment, the sample is cultured in a serum-free medium. Such a serum-free medium may also include a serum substitute medium, the serum substitute being based on chemically defined components to avoid the use of human or animal-derived serum. In one embodiment, the medium does not contain any animal-derived products. In an alternative embodiment, the sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)).

[0108] The culture medium may further contain other components that can help the growth and proliferation of γδT cells. Examples of other components that may be added include, but are not limited to, plasma or serum, purified proteins such as albumin, lipid sources such as low-density lipoprotein (LDL), vitamins, amino acids, steroids, and any other nutritional supplements that assist or promote cell proliferation and / or survival.

[0109] Antibodies or fragments thereof Isolated and specifically binds to the gamma variable 4 (Vγ4) chain of the γδ T cell receptor (TCR). Antibodies or fragments thereof are provided herein. In particular, the antibodies or fragments thereof do not bind to (or cross-react with) the gamma variable 2 (Vγ2) chain of the γδTCR. It should be understood that this is in relation to the Vγ4 and Vγ2 chains from the same species. Preferably, the species is Homo sapiens (human), and therefore the antibodies or fragments thereof can specifically bind to the human gamma variable 4 (Vγ4) chain of the γδT cell receptor (TCR) and not to the human gamma variable 2 (Vγ2) chain of the γδTCR. For example, the human Vγ4 chain may have the sequence of amino acids 1-99 of SEQ ID NO: 1, and / or the human Vγ2 chain may have the sequence of SEQ ID NO: 335. In other species, the antibodies or fragments thereof specifically bind to the species-specific ortholog of the human gamma variable 4 (Vγ4) chain of the γδT cell receptor (TCR) and not to the species-specific ortholog of the human gamma variable 2 (Vγ2) chain of the γδTCR. Therefore, the antibody or fragment may specifically bind to the human gamma variable 4 (Vγ4) chain of the γδ T cell receptor (TCR) or its non-human orthologue having a sequence corresponding to amino acids 1-99 of SEQ ID NO: 1, but may not bind to the human gamma variable 2 (Vγ2) chain of the γδ TCR or its non-human orthologue having a sequence corresponding to SEQ ID NO: 335. In this context, an orthologue may mean a gamma chain sequence that has the highest sequence similarity to the reference sequence, or preferably one that has the same function (e.g., interaction with orthologous congener ligands in vivo). For example, in mice, the protein designated as Vγ7 under Heilig & Tonegave nomenclature is functionally most closely related to human Vγ4 (Barros et al. (2016) Cell, 167:203-218.e17).

[0110] This development is profound. For example, in humans, the Vγ4 and Vγ2 chains share 91% sequence identity (only nine amino acids differ). Therefore, it is difficult to obtain an antibody that binds to (human) Vγ4 but not to (human) Vγ2, and it was not anticipated in the art that such an antibody could be created.

[0111] When referring to an antibody or fragment that specifically binds to the Vγ4 chain of the γδTCR, this generally means that the binding of the antibody or fragment to the Vγ4 chain is statistically significantly increased compared to the negative control antibody and / or negative control antigen (e.g., as measured via binding in an ELISA assay, optionally a DELFIA ELISA assay, or SPR). The level detected relative to the negative control antibody and / or negative control antigen can be considered the background level of the assay used, representing "noise" in the assay system, as will be well understood by those skilled in the art. In certain embodiments, a signal level exceeding a predetermined threshold relative to the background level can be considered to represent the detection of binding (e.g., about 1, 2, 3, 4, 5 or more times the background level). For example, in a DELFIA ELISA assay, a signal level of 5 or more times the background level can be considered to indicate antibody binding to the antigen. Those skilled in the art can determine appropriate thresholds based on the assay system being used. Conversely, when referring to an antibody or fragment that does not bind to (or cross-react with) the Vγ2 chain of the γδTCR, this generally means that the binding of the antibody or fragment to the Vγ2 chain is not statistically significantly increased compared to the negative control antibody and / or negative control antigen (as measured, for example, via binding in an ELISA assay, optionally a DELFIA ELISA assay, or SPR). This is shown, for example, in Figure 2A and explained in Example 4. According to all aspects and embodiments of the present invention disclosed herein, this property can also be expressed as a difference in magnification change in the binding level detected between the antibody or fragment and the Vγ4 chain versus the antibody or fragment and the Vγ2 chain (as measured, for example, via binding in an ELISA assay, optionally a DELFIA ELISA assay, or SPR). For example, the antibody or fragment shows at least about 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 15-fold increase in binding to the Vγ4 chain compared to binding to the Vγ2 chain. Increases of 0x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10000x, 15000x, 25000x, 50000x, 75000x, and 95000x or more can be observed. This is shown, for example, in Figure 2B and explained in Example 4. However, such doubling is considered conservative because it is calculated assuming that all Vγ2 signals exceeding the control are not background noise. However, as mentioned above, those skilled in the art can instead exclude low signals above the background level in such a DELFIA ELISA assay as assay noise (e.g., approximately 1, 2, 3, 4, 5 times or more the background level), and signals below these thresholds can be considered nonspecific background binding.

[0112] In one embodiment, the antibody or fragment is an scFv, Fab, Fab', F(ab')2, Fv, a variable domain (e.g., VH or VL), a diabody, a minibody, or a monoclonal antibody. In a particular embodiment, the antibody or fragment is an scFv. In another particular embodiment, the antibody is a monoclonal antibody.

[0113] The antibodies described herein may be of any type, e.g., IgG, IgA, IgM, IgE, IgD or isotypes thereof, and may contain kappa light chains or lambda light chains. In one embodiment, the antibody is an IgG antibody, e.g., at least one of isotypes, IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody is IgG1. In further embodiments, the antibody may be in the form of an IgG type modified to give desired properties, such as having a mutated Fc to reduce effector function, extend half-life, alter ADCC, or improve hinge stability. Such modifications are well known in the art, and exemplary embodiments are described herein. For example, the antibody or a fragment thereof may contain an IgG1 constant domain comprising the amino acid sequence by SEQ ID NO: 332 or SEQ ID NO: 333.

[0114] In one embodiment, the antibody or fragment is human. Therefore, the antibody or fragment may be derived from a human immunoglobulin (Ig) sequence. The CDR, framework, and / or constant region of the antibody (or fragment) may be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework, and / or constant region may be substantially identical with respect to a human Ig sequence, particularly a human IgG sequence. The advantage of using a human antibody is that it has low or no immunogenicity in humans.

[0115] Antibodies or fragments thereof may also be chimeras, such as mouse-human antibody chimeras.

[0116] Alternatively, the antibody or its fragment may be derived from a non-human species, such as a mouse. Such non-human antibodies can be modified to increase their similarity to antibody variants naturally produced in humans, and thus the antibody or its fragment may be partially or completely humanized. In one embodiment, the antibody or its fragment is therefore humanized.

[0117] Antibodies that target epitopes Antibodies (or fragments thereof) that bind to the epitope on the Vγ4 chain of γδTCR are provided herein. Binding to the epitope on the Vγ4 chain may optionally affect γδTCR activity, such as activation or inhibition. The antibodies (or fragments thereof) may have a blocking effect by preventing the binding or interaction of another antibody or molecule. The antibodies are specific to the Vγ4 chain of γδTCR and do not bind to epitopes of other antigens, such as the Vγ2 chain or the Vγ8 chain of γδTCR, as defined herein.

[0118] In one embodiment, the epitope may be an activating epitope for γδT cells. An "activating" epitope may include, for example, the modulation of TCR-related functions such as TCR downregulation, cell degranulation, cytotoxicity, proliferation, recruitment, prolongation of survival or resistance to depletion, intracellular signaling, cytokine or growth factor secretion, phenotypic changes, or altered gene expression. For example, binding of an activating epitope may stimulate the expansion (i.e., proliferation) of a γδT cell population, preferably a Vγ4+ T cell population. Therefore, these antibodies can be used to modulate γδT cell activation and thereby modulate the immune response. Thus, in one embodiment, binding of an activating epitope downregulates the γδTCR. In additional or alternative embodiments, binding of an activating epitope activates γδT cell degranulation. In further additional or alternative embodiments, binding of an activating epitope activates γδT cells to kill target cells (e.g., cancer cells).

[0119] In one embodiment, an antibody or fragment thereof blocks Vγ4 and inhibits TCR binding (e.g., via steric hindrance). By blocking Vγ4, the antibody can inhibit TCR activation and / or signaling. Thus, the epitope may be an inhibitory epitope for γδ T cells. An "inhibitory" epitope may include, for example, blocking TCR function and thereby suppressing TCR activation.

[0120] The epitope preferably consists of at least one extracellular, soluble, hydrophilic, external, or cytoplasmic portion of the Vγ4 chain of the γδTCR.

[0121] In certain embodiments, the epitope does not include the epitope found in the non-germ-encoding region of the Vγ4 chain of the γδTCR, particularly in the CDR3 of the Vγ4 chain. In preferred embodiments, the epitope is located within the framework region of the Vγ4 chain of the γδTCR, which may be the hypervariable 4 region of framework region 3. It will be understood that such binding allows for specific recognition of the entire Vγ4 chain without being limited by the highly variable TCR sequence (particularly CDR3) between Vγ4 chains. Therefore, it will be understood that any γδTCR containing any Vγ4 chain can be recognized using the antibody or fragment thereof as defined herein, regardless of the specificity of the γδTCR.

[0122] The γδ receptor can bind to various regulatory ligands independently and via spatially distinct domains. Consistent with such multimode ligand binding, a recent study by Melandri et al. (2018) Nat. Immunol. 19:1352-1365 shows that the binding of the human TCR to the endogenous BTNL3 ligand is via a distinct domain located at the N-terminus of CDR3 on the γ4 chain. The authors emphasize that because BTNL3 binding is mediated through this specific germline region of the TCR, the more C-terminal somatic recombinant CDR3 loop can freely bind to other ligands independently. Furthermore, this sub-region of framework region 3 (FR3), also referred to as "hypervariable region 4" (HV4), differs from the human γ2 chain by four amino acids. However, no specific anti-Vγ4 antibody is disclosed by Melandri et al., nor is it suggested how such an antibody could be obtained. In fact, the prevailing view was that this was impossible due to the significant sequence homology (91% sequence identity) shared between human Vγ4 and human Vγ2 chains.

[0123] Antibodies that bind within the HV4 region may still allow binding to the CDR3 region of the γ4 chain, with the added advantage of providing a conjugate that is more specific to γ4 than to γ2. Furthermore, since HV4 is encoded in the germline, some antibodies targeting this region may recognize all Vγ4 chains, while other antibodies that recognize Vγ4 may be specific to a particular Vγ4 chain.

[0124] This disclosure provides antibodies and fragments thereof that can specifically bind to the HV4 region of the Vγ4 chain. Accordingly, in one embodiment, the antibody or fragment binds to the epitope of the HV4 region of the Vγ4 chain. The HV4 region includes amino acids 67-82 of SEQ ID NO: 1. Accordingly, in one embodiment, the epitope includes one or more amino acid residues within amino acid region 67-82 of SEQ ID NO: 1, e.g., a portion of the Vγ4 chain that is not part of the CDR1, CDR2 and / or CDR3 sequences. By doing so, the antibody or fragment can modulate the interaction between Vγ4+TCR and BTNL3 / 8. In one embodiment, the epitope does not include amino acid residues within amino acid region 96-106 (CDR3) of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues within amino acid region 50-57 (CDR2) of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues within amino acid region 27-32 (CDR1) of SEQ ID NO: 1.

[0125] In certain embodiments, an antibody or fragment thereof can activate the Vγ4+ TCR when bound to one or more amino acids 67-82 of SEQ ID NO: 1.

[0126] Similar to well-characterized αβT cells, γδT cells utilize different sets of somatically reconstituted variable (V), diversity (D) (β and δ only), binding (J), and stationary (C) genes, although γδT cells contain fewer V, D, and J segments than αβT cells. In one embodiment, the epitope bound by the antibody (or fragment thereof) does not include the epitope found in the J region of the Vγ4 chain. Therefore, the antibody or fragment can bind only to the V region of the Vγ4 chain. Thus, in one embodiment, the epitope consists of the epitope in the V region of the γδTCR (e.g., amino acid residues 1-99 of SEQ ID NO: 1).

[0127] References to the epitope are made in relation to Luoma et al. (2013) Immunity39:1032-1042 and the Vγ4 sequence described in the RCSB Protein Databank entry:4MNH, indicated as Sequence ID No. 1. SSNLEGRTKSVIRQTGSSAEITCDLAEGSTGYIHWYLHQEGKAPQRLLYYDSYTSSVVLESGISPGKYDTYGSTRKNLRMILRNLIENDSGVYYCATWDEKYYKKLFGSGTTLVVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADSRGGLEVLFQ(Sequence ID 1)

[0128] Sequence ID 1 represents a soluble TCR containing the V region (also called the variable domain) and the J region. The V region contains amino acid residues 1-99, the J region contains amino acid residues 102-116, and the constant region from TCRβ contains amino acid residues 117-256. Within the V region, CDR1 is defined as amino acid residues 27-32 of Sequence ID 1, CDR2 is defined as amino acid residues 50-57 of Sequence ID 1, and CDR3 is defined as amino acid residues 96-106 of Sequence ID 1.

[0129] The inventors have confirmed that amino acids K76 (i.e., lysine at position 76) and M80 (i.e., methionine at position 80) of SEQ ID NO: 1 may be particularly important for binding to the HV4 region of the (human) Vγ4 chain (Example 6). Therefore, the epitope may or may consist of K76 and / or M80 of SEQ ID NO: 1.

[0130] The inventors have further confirmed that amino acids within the amino acid region 71-79 of SEQ ID NO: 1 may be particularly important for binding to the HV4 region of the (human) Vγ4 chain. Therefore, in a further embodiment, the epitope is one or more within the amino acid region 71-79 of SEQ ID NO: 1. Contains amino acid residues.

[0131] In one embodiment, the epitope contains one or more amino acid residues within the described region, such as two, three, four, five, six, seven, eight, nine, or ten or more.

[0132] In one embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within the amino acid region 67-82 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more (e.g., three or more, e.g., five or more) amino acid residues within the amino acid region 71-79 of SEQ ID NO: 1.

[0133] It will be further understood that the antibody (or fragment thereof) does not need to bind to all amino acids within a defined range. Such an epitope may be called a linear epitope. For example, an antibody that binds to an epitope containing amino acid residues within the amino acid region 67-82 of SEQ ID NO: 1 may bind only to one or more amino acid residues at both ends of the range (i.e., amino acids 67 and 82), including, for example, amino acids within the range of choice (i.e., amino acids 71, 73, 75, 76, and 79).

[0134] For example, the inventors have found that amino acid residues 71, 73, 75, 76, and 79 of SEQ ID NO: 1 can form an epitope to which an anti-Vγ4 antibody or a fragment thereof can bind (Example 8). Thus, in one embodiment, the epitope comprises at least one of the amino acid residues 71, 73, 75, 76, and 79 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one, two, three, four, or five (particularly four or five) amino acids selected from amino acid residues 71, 73, 75, 76, and 79 of SEQ ID NO: 1.

[0135] In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 67-82 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 71-79 of SEQ ID NO: 1.

[0136] In a further embodiment, the epitope comprises amino acid residues 71-79 of SEQ ID NO: 1, or preferably consists of amino acid residues 71-79 of SEQ ID NO: 1. In yet another embodiment, the epitope comprises amino acid residues 71, 73, 75, 76 and 79 of SEQ ID NO: 1, or preferably consists of amino acid residues 71, 73, 75, 76 and 79 of SEQ ID NO: 1.

[0137] Various techniques are known in the art for determining which epitopes are bound by antibodies. Exemplary techniques include, for example, routine cross-blocking assays, alanine scanning mutation analysis, peptide blot analysis, peptide cleavage analysis, crystallographic experiments, and NMR analysis. Furthermore, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used. Another method that can be used to identify amino acids in polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry (as described in Example 8). In general terms, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex then moves to water, and the exchangeable protons in the amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons in the amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit a relatively higher mass compared to amino acids that are not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts.

[0138] Furthermore, or alternatively, antigen chimera and mutagenesis experiments can be used to identify the amino acids in the polypeptide with which the antibody interacts (as described in Example 6). In general terms, this method involves creating a series of one or more chimeric antigens, where the amino acid sequence of the first reference antigen may be systematically modified based on the amino acid sequence of the second reference antigen to replace one or more amino acids of the first reference antigen with corresponding amino acids from the second reference antigen. In this context, “corresponding amino acids” means the amino acids at equivalent positions in the sequences of the first and second reference antigens when their sequences are aligned. The binding of the test antibody to each of the first reference antigen, the second reference antigen, and / or the series of one or more chimeric antigens is then measured. The loss / gain of binding to each antigen may then be due to specific amino acid changes made to the first and / or second reference sequences. Whether the antibody can bind to the first and / or second reference antigen may already be known. For example, as described in Example 6, the first reference antigen may be a human Vγ4 chain, the second reference antigen may be a human Vγ2 chain, and a series of chimeric antigens are prepared by substituting one or more amino acids in the Vγ4 chain with one or more amino acids in the Vγ2 chain sequence.

[0139] antibody sequence Anti-Vγ4 antibodies or fragments thereof may be described with reference to their CDR sequences.

[0140] Therefore, in one embodiment, the anti-Vγ4 antibody or a fragment thereof is CDR3 containing one of sequence numbers 2 to 47, preferably a sequence having at least 80% sequence identity with sequence number 10 and / or sequence number 33, CDR2 includes one of sequence numbers 48-70 and sequences A1-A23 (in Figure 1), preferably sequence number 56 and / or A9, having at least 80% sequence identity, and / or The CDR1 comprises one or more sequences having at least 80% sequence identity with any one of sequence numbers 71 to 116, preferably sequence number 79 and / or sequence number 102.

[0141] In one embodiment, the anti-Vγ4 antibody or fragment thereof includes CDR3, which contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs. 2 to 47. In one embodiment, the antibody or fragment thereof includes CDR2, which contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs. 48 to 70 and sequences A1 to A23 (in Figure 1). In one embodiment, the antibody or fragment thereof includes CDR1, which contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs. 71 to 116.

[0142] In some embodiments, the anti-Vγ4 antibody or a fragment thereof A heavy chain CDR3 (HCDR3) containing one of sequence numbers 2 to 24, preferably a sequence having at least 80% sequence identity with sequence number 10, A heavy chain CDR2 (HCDR2) containing one of sequence numbers 48 to 70, preferably a sequence having at least 80% sequence identity with sequence number 56, and / or It may contain one or more heavy chain CDR1 (HCDR1) sequences, preferably one of sequence numbers 71 to 93, which have at least 80% sequence identity with sequence number 79.

[0143] Alternatively, an anti-Vγ4 antibody or a fragment thereof may be used. A light chain CDR3 (LCDR3) containing one of sequence numbers 25 to 47, preferably a sequence having at least 80% sequence identity with sequence number 33, A light chain CDR2 (LCDR2) containing any one of sequences A1 to A23 (in Figure 1), preferably a sequence having at least 80% sequence identity with sequence number A9, and / or One of sequence numbers 94-116, preferably sequence number 102 and at least 80% It may contain one or more light chain CDR1 (LCDR1) sequences that have sequence identity.

[0144] In one embodiment, the antibody or fragment contains a CDR3 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 2 to 47. In one embodiment, the antibody or fragment contains a CDR2 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 48 to 70 and sequences A1 to A23 (in Figure 1). In one embodiment, the antibody or fragment contains a CDR1 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 71 to 116.

[0145] In one embodiment, the antibody or fragment comprises CDR3, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 2 to 47. 3. In one embodiment, the antibody or fragment comprises CDR2, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 48 to 70 and sequences A1 to A23 (in Figure 1). 4. In one embodiment, the antibody or fragment comprises CDR1, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 71 to 116.

[0146] In one embodiment, the antibody or fragment comprises a VH region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 24, and / or a VL region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 25 to 47.

[0147] In one embodiment, the antibody or fragment comprises a VH region containing a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 24, and / or a VL region containing a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 25 to 47.

[0148] In one embodiment, the antibody or fragment comprises a VH region containing a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 2 to 24, and / or a VL region containing a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 25 to 47.

[0149] In one embodiment, the antibody or fragment comprises a VH region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 24, and a VL region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 25 to 47. In one embodiment, the antibody or fragment comprises a VH region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 24. The region includes a VL region containing a CDR3 that has at least 80% sequence identity with one of sequence numbers 25-47.

[0150] Embodiments in this specification that refer to “at least 80%” or “80% or more” will be understood to include all values ​​of sequence identity of 80% or more, such as 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In one embodiment, the antibody or fragment contains at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with respect to a given sequence.

[0151] Instead of a percentage of sequence identity, embodiments may also be defined by one or more amino acid changes, e.g., one or more additions, substitutions, and / or deletions. In one embodiment, a sequence may contain up to five amino acid changes, e.g., up to three amino acid changes, and in particular up to two amino acid changes. For example, a sequence may contain up to five amino acid substitutions, e.g., up to three amino acid substitutions, and in particular up to one or two amino acid substitutions. For example, the CDR3 of an antibody or fragment may contain, or more preferably consist of, a sequence having two or fewer, more preferably one or fewer, substitutions compared to any one of sequence numbers 2-47.

[0152] Appropriately, any residue in CDR1, CDR2, or CDR3 that is different from the corresponding residues in SEQ ID NOs. 2-116 and sequences A1-A23 is a conserved substitution with respect to its corresponding residue. For example, any residue in CDR3 that is different from the corresponding residues in SEQ ID NOs. 2-47 is a conserved substitution with respect to its corresponding residue.

[0153] In one embodiment, the antibody or a fragment thereof (i) A VH region containing a CDR3 that has at least 80% sequence identity with any one of sequence numbers 2 to 24, (ii) A VH region containing a CDR2 having at least 80% sequence identity with any one of sequence numbers 48 to 70, (iii) A VH region containing CDR1 that has at least 80% sequence identity with any one of sequence numbers 71 to 93, (iv) A VL region containing a CDR3 containing a sequence having at least 80% sequence identity with any one of sequence numbers 25-47, (v) A VL region including CDR2 containing a sequence having at least 80% sequence identity with any one of sequences A1 to A23 (Figure 1), and / or (vi) A VL region containing a CDR1 containing a sequence having at least 80% sequence identity with any one of sequence numbers 94-116.

[0154] In one embodiment, the antibody or a fragment thereof (i) A VH region containing a CDR3 that has at least 80% sequence identity with any one of sequence numbers 2 to 24, (ii) A VH region including a CDR2 containing a sequence having at least 80% sequence identity with any one of sequence numbers 48 to 70, and (iii) A heavy chain comprising a VH region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 71 to 93.

[0155] In one embodiment, the antibody or a fragment thereof (i) A VL region containing a CDR3 having at least 80% sequence identity with any one of sequence numbers 25-47, (ii) A VL region including CDR2 containing a sequence having at least 80% sequence identity with any one of sequences A1 to A23 (Figure 1), and (iii) Having at least 80% sequence identity with any one of sequence numbers 94-116 It includes a light chain comprising a VL region containing a CDR1 containing the sequence.

[0156] In one embodiment, the antibody or fragment comprises (or consists of) a VH region comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of sequence numbers 2-24, such as sequence numbers 10, 4, 14, 15, 17, 19, or 23. In one embodiment, the antibody or fragment comprises (or consists of) a VH region comprising a CDR2 containing a sequence having at least 80% sequence identity with any one of sequence numbers 48-70, such as sequence numbers 56, 50, 60, 61, 63, 65, or 69. In one embodiment, the antibody or fragment comprises (or consists of) a VH region comprising a CDR1 containing a sequence having at least 80% sequence identity with any one of sequence numbers 71-93, such as sequence numbers 79, 73, 83, 84, 86, 88, or 92.

[0157] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 10, CDR2 containing the sequence of sequence number 56, and CDR1 containing the sequence of sequence number 79. In one embodiment, CDR3 consists of the sequence of sequence number 10, CDR2 consists of the sequence of sequence number 56, and CDR1 consists of the sequence of sequence number 79.

[0158] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 4, CDR2 containing the sequence of sequence number 50, and CDR1 containing the sequence of sequence number 73. In one embodiment, CDR3 consists of the sequence of sequence number 4, CDR2 consists of the sequence of sequence number 50, and CDR1 consists of the sequence of sequence number 73.

[0159] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 14, CDR2 containing the sequence of sequence number 60, and CDR1 containing the sequence of sequence number 83. In one embodiment, CDR3 consists of the sequence of sequence number 14, CDR2 consists of the sequence of sequence number 60, and CDR1 consists of the sequence of sequence number 83.

[0160] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 15, CDR2 containing the sequence of sequence number 61, and CDR1 containing the sequence of sequence number 84. In one embodiment, CDR3 consists of the sequence of sequence number 15, CDR2 consists of the sequence of sequence number 61, and CDR1 consists of the sequence of sequence number 84.

[0161] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 17, CDR2 containing the sequence of sequence number 63, and CDR1 containing the sequence of sequence number 86. In one embodiment, CDR3 consists of the sequence of sequence number 17, CDR2 consists of the sequence of sequence number 63, and CDR1 consists of the sequence of sequence number 86.

[0162] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 19, CDR2 containing the sequence of sequence number 65, and CDR1 containing the sequence of sequence number 88. In one embodiment, CDR3 consists of the sequence of sequence number 19, CDR2 consists of the sequence of sequence number 65, and CDR1 consists of the sequence of sequence number 88.

[0163] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 23, CDR2 containing the sequence of sequence number 69, and CDR1 containing the sequence of sequence number 92. In one embodiment, CDR3 consists of the sequence of sequence number 23, CDR2 consists of the sequence of sequence number 69, and CDR1 consists of the sequence of sequence number 92.

[0164] In one embodiment, the antibody or fragment thereof includes (or consists of) a VL region containing a CDR3 having at least 80% sequence identity with one of sequence numbers 25-47, such as sequence numbers 33, 27, 37, 38, 40, 42, or 46. Morphologically, the antibody or its fragment includes (or consists of) a VL region containing a CDR2 that has at least 80% sequence identity with any one of sequences A1-A23 (in Figure 1), such as sequence A9, A3, A13, A14, A16, A18, or A22. In one embodiment, the antibody or its fragment includes (or consists of) a VL region containing a CDR1 that has at least 80% sequence identity with any one of sequence numbers 94-116, such as sequence numbers 102, 96, 106, 107, 109, 111, or 115.

[0165] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 33, CDR2 containing the sequence of sequence A9, and CDR1 containing the sequence of sequence number 102. In one embodiment, CDR3 consists of the sequence of sequence number 33, CDR2 consists of the sequence of sequence A9, and CDR1 consists of the sequence of sequence number 102.

[0166] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 27, CDR2 containing the sequence of sequence A3, and CDR1 containing the sequence of sequence number 96. In one embodiment, CDR3 consists of the sequence of sequence number 27, CDR2 consists of the sequence of sequence A3, and CDR1 consists of the sequence of sequence number 96.

[0167] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 37, CDR2 containing the sequence of sequence A13, and CDR1 containing the sequence of sequence number 106. In one embodiment, CDR3 consists of the sequence of sequence number 37, CDR2 consists of the sequence of sequence A13, and CDR1 consists of the sequence of sequence number 106.

[0168] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 38, CDR2 containing the sequence of sequence A14, and CDR1 containing the sequence of sequence number 107. In one embodiment, CDR3 consists of the sequence of sequence number 38, CDR2 consists of the sequence of sequence A14, and CDR1 consists of the sequence of sequence number 107.

[0169] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 40, CDR2 containing the sequence of sequence A16, and CDR1 containing the sequence of sequence number 109. In one embodiment, CDR3 consists of the sequence of sequence number 40, CDR2 consists of the sequence of sequence A16, and CDR1 consists of the sequence of sequence number 109.

[0170] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 42, CDR2 containing the sequence of sequence A18, and CDR1 containing the sequence of sequence number 111. In one embodiment, CDR3 consists of the sequence of sequence number 42, CDR2 consists of the sequence of sequence A18, and CDR1 consists of the sequence of sequence number 111.

[0171] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 46, CDR2 containing the sequence of sequence A22, and CDR1 containing the sequence of sequence number 115. In one embodiment, CDR3 consists of the sequence of sequence number 46, CDR2 consists of the sequence of sequence A22, and CDR1 consists of the sequence of sequence number 115.

[0172] In one embodiment, the antibody or fragment comprises one or more CDR sequences as shown in Figure 1. In a further embodiment, the antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1140_P01_G08[G4_12] or clone 1139_P01_A04[G4_03] as shown in Figure 1.

[0173] Therefore, the anti-Vγ4 antibody or its fragments are (a) HCDR1 having sequence number 79, HCDR2 having sequence number 56 and sequence number A VH comprising HCDR3 having number 10, wherein the VH optionally comprises or consists of sequence number 125. A VL comprising LCDR1 having sequence number 102, LCDR2 having sequence number A9 (Figure 1), and LCDR3 having sequence number 33, wherein the VL optionally includes or consists of sequence number 148. (b) A VH comprising HCDR1 having sequence number 86, HCDR2 having sequence number 63, and HCDR3 having sequence number 17, wherein the VH optionally comprises or includes sequence number 132, A VL comprising LCDR1 having sequence number 109, LCDR2 having sequence number A16 (Figure 1), and LCDR3 having sequence number 40, wherein the VL optionally includes or consists of sequence number 155. (c) A VH comprising HCDR1 having sequence number 73, HCDR2 having sequence number 50, and HCDR3 having sequence number 4, wherein the VH optionally comprises or consists of sequence number 119. A VL comprising LCDR1 having sequence number 96, LCDR2 having sequence number A3 (Figure 1), and LCDR3 having sequence number 27, wherein the VL optionally includes or consists of sequence number 142. (d) A VH comprising HCDR1 having sequence number 83, HCDR2 having sequence number 60, and HCDR3 having sequence number 14, wherein the VH optionally comprises or consists of sequence number 129. A VL comprising LCDR1 having sequence number 106, LCDR2 having sequence number A13 (Figure 1), and LCDR3 having sequence number 37, wherein the VL optionally includes or consists of sequence number 152. (e) A VH comprising HCDR1 having sequence number 84, HCDR2 having sequence number 61, and HCDR3 having sequence number 15, wherein the VH optionally comprises or consists of sequence number 130. A VL comprising LCDR1 having SEQ ID NO: 107, LCDR2 having SEQ A14 (of FIG. 1), and LCDR3 having SEQ ID NO: 38, and optionally, said VL comprises or consists of SEQ ID NO: 153, (f) A VH comprising HCDR1 having SEQ ID NO: 88, HCDR2 having SEQ ID NO: 65, and HCDR3 having SEQ ID NO: 19, and optionally, said VH comprises or consists of SEQ ID NO: 134, A VL comprising LCDR1 having SEQ ID NO: 111, LCDR2 having SEQ A18 (of FIG. 1), and LCDR3 having SEQ ID NO: 42, and optionally, said VL comprises or consists of SEQ ID NO: 157, (g) A VH comprising HCDR1 having SEQ ID NO: 92, HCDR2 having SEQ ID NO: 69, and HCDR3 having SEQ ID NO: 23, and optionally, said VH comprises or consists of SEQ ID NO: 138, A VL comprising LCDR1 having SEQ ID NO: 115, LCDR2 having SEQ A22 (of FIG. 1), and LCDR3 having SEQ ID NO: 46, and optionally, said VL comprises or consists of SEQ ID NO: 161, (h) A VH comprising HCDR1 having SEQ ID NO: 71, HCDR2 having SEQ ID NO: 48, and HCDR3 having SEQ ID NO: 2, and optionally, said VH comprises or consists of SEQ ID NO: 117, A VL comprising LCDR1 having SEQ ID NO: 94, LCDR2 having SEQ A1 (of FIG. 1), and LCDR3 having SEQ ID NO: 25, and optionally, said VL comprises or consists of SEQ ID NO: 140, (i) A VH comprising HCDR1 having SEQ ID NO: 72, HCDR2 having SEQ ID NO: 49, and HCDR3 having SEQ ID NO: 3, and optionally, said VH comprises or consists of SEQ ID NO: 118, A VL comprising LCDR1 having SEQ ID NO: 95, LCDR2 having SEQ A2 (of FIG. 1), and LCDR3 having SEQ ID NO: 26, and optionally, said VL comprises or consists of SEQ ID NO: 141, (j) A VH comprising HCDR1 having SEQ ID NO: 74, HCDR2 having SEQ ID NO: 51, and HCDR3 having SEQ ID NO: 5, wherein optionally the VH comprises or consists of a VH comprising SEQ ID NO: 120, A VL comprising LCDR1 having SEQ ID NO: 97, LCDR2 having SEQ A4 (of FIG. 1), and LCDR3 having SEQ ID NO: 28, wherein optionally the VL comprises or consists of a VL comprising SEQ ID NO: 143, (k) A VH comprising HCDR1 having SEQ ID NO: 75, HCDR2 having SEQ ID NO: 52, and HCDR3 having SEQ ID NO: 6, wherein optionally the VH comprises or consists of a VH comprising SEQ ID NO: 121, A VL comprising LCDR1 having SEQ ID NO: 98, LCDR2 having SEQ A5 (of FIG. 1), and LCDR3 having SEQ ID NO: 29, wherein optionally the VL comprises or consists of a VL comprising SEQ ID NO: 144, (l) A VH comprising HCDR1 having SEQ ID NO: 76, HCDR2 having SEQ ID NO: 53, and HCDR3 having SEQ ID NO: 7, wherein optionally the VH comprises or consists of a VH comprising SEQ ID NO: 122, A VL comprising LCDR1 having SEQ ID NO: 99, LCDR2 having SEQ A6 (of FIG. 1), and LCDR3 having SEQ ID NO: 30, wherein optionally the VL comprises or consists of a VL comprising SEQ ID NO: 145, (m) A VH comprising HCDR1 having SEQ ID NO: 77, HCDR2 having SEQ ID NO: 54, and HCDR3 having SEQ ID NO: 8, wherein optionally the VH comprises or consists of a VH comprising SEQ ID NO: 123, A VL comprising LCDR1 having SEQ ID NO: 100, LCDR2 having SEQ A7 (of FIG. 1), and LCDR3 having SEQ ID NO: 31, wherein optionally the VL comprises or consists of a VL comprising SEQ ID NO: 146, (n) A VH comprising HCDR1 having SEQ ID NO: 78, HCDR2 having SEQ ID NO: 55, and HCDR3 having SEQ ID NO: 9, wherein optionally the VH comprises or consists of a VH comprising SEQ ID NO: 124, A VL comprising LCDR1 having sequence number 101, LCDR2 having sequence number A8 (Figure 1), and LCDR3 having sequence number 32, wherein the VL optionally includes or consists of sequence number 147. (o) A VH comprising HCDR1 having sequence number 80, HCDR2 having sequence number 57, and HCDR3 having sequence number 11, wherein the VH optionally comprises or consists of sequence number 126. A VL comprising LCDR1 having sequence number 103, LCDR2 having sequence number A10 (Figure 1), and LCDR3 having sequence number 34, wherein the VL optionally includes or consists of sequence number 149. (p) A VH comprising HCDR1 having sequence number 81, HCDR2 having sequence number 58, and HCDR3 having sequence number 12, wherein the VH optionally comprises or consists of sequence number 127. A VL comprising LCDR1 having sequence number 104, LCDR2 having sequence number A11 (Figure 1), and LCDR3 having sequence number 35, wherein the VL optionally includes or consists of sequence number 150. (q) A VH comprising HCDR1 having sequence number 82, HCDR2 having sequence number 59, and HCDR3 having sequence number 13, wherein the VH optionally comprises or consists of sequence number 128. A VL comprising LCDR1 having sequence number 105, LCDR2 having sequence number A12 (Figure 1), and LCDR3 having sequence number 36, wherein the VL optionally has sequence number 15 A VL containing or consisting of 1, (r) A VH comprising HCDR1 having sequence number 85, HCDR2 having sequence number 62, and HCDR3 having sequence number 16, wherein the VH optionally comprises or consists of sequence number 131. A VL comprising LCDR1 having sequence number 108, LCDR2 having sequence number A15 (Figure 1), and LCDR3 having sequence number 39, wherein the VL optionally includes or consists of sequence number 154. (s) A VH comprising HCDR1 having sequence number 87, HCDR2 having sequence number 64, and HCDR3 having sequence number 18, wherein the VH optionally comprises or consists of sequence number 133. A VL comprising LCDR1 having sequence number 110, LCDR2 having sequence number A17 (in Figure 1), and LCDR3 having sequence number 41, wherein the VL optionally includes or consists of sequence number 156. (t) A VH comprising HCDR1 having sequence number 89, HCDR2 having sequence number 66, and HCDR3 having sequence number 20, wherein the VH optionally comprises or consists of sequence number 135. A VL comprising LCDR1 having sequence number 112, LCDR2 having sequence number A19 (Figure 1), and LCDR3 having sequence number 43, wherein the VL optionally includes or consists of sequence number 158. (u) A VH comprising HCDR1 having sequence number 90, HCDR2 having sequence number 67, and HCDR3 having sequence number 21, wherein the VH optionally comprises or consists of sequence number 136. A VL comprising LCDR1 having sequence number 113, LCDR2 having sequence number A20 (Figure 1), and LCDR3 having sequence number 44, wherein the VL optionally includes or consists of sequence number 159. (v) A VH comprising HCDR1 having sequence number 91, HCDR2 having sequence number 68, and HCDR3 having sequence number 22, wherein the VH optionally comprises or consists of sequence number 137. A VL comprising LCDR1 having sequence number 114, LCDR2 having sequence number A21 (Figure 1), and LCDR3 having sequence number 45, wherein the VL optionally includes or consists of sequence number 160. and / or, (w) A VH comprising HCDR1 having sequence number 93, HCDR2 having sequence number 70, and HCDR3 having sequence number 24, wherein the VH optionally comprises or consists of sequence number 139. A VL containing LCDR1 having sequence number 116, LCDR2 having sequence number A23 (as shown in Figure 1), and LCDR3 having sequence number 47, wherein the VL may optionally contain one or more VLs that include or consist of sequence number 162.

[0174] Appropriately, the VH region and VL region described above each include four framework regions (FR1 to FR4). In one embodiment, the antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that has at least 80% sequence identity with any one of the framework regions of SEQ ID NOs. 117 to 162. In one embodiment, the antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that has at least 90% sequence identity, such as at least 95%, 97%, or 99%, with any one of the framework regions of SEQ ID NOs. 117 to 162. In one embodiment, the antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that includes any one of the sequences of SEQ ID NOs. 117 to 162. In one embodiment, the antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) consisting of any one of the sequences of SEQ ID NOs. 117 to 162. Includes 1, FR2, FR3 and / or FR4).

[0175] The antibodies described herein may be defined by their complete light chain variable sequence and / or heavy chain variable sequence. Thus, in one embodiment, an anti-Vγ4 antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 117-162.

[0176] In one embodiment, the antibody or fragment includes a VH region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 117-139. In another embodiment, the antibody or fragment includes a VH region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 117-139. In a further embodiment, the VH region includes an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 125, 119, 129, 130, 132, 134, or 138. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 125, 119, 129, 130, 132, 134, or 138.

[0177] In one embodiment, the antibody or fragment includes a VL region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 140-162. In another embodiment, the antibody or fragment includes a VL region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 140-162. In a further embodiment, the VL region includes an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 148, 142, 152, 153, 155, 157, or 161. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 148, 142, 152, 153, 155, 157, or 161.

[0178] In a further embodiment, the antibody or fragment comprises a VH region containing an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 117-139, and a VL region containing an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 140-162.

[0179] In one embodiment, the antibody or fragment includes a VH region containing the amino acid sequence of SEQ ID NO: 125 (1140_P01_G08)[G4_12]. In one embodiment, the antibody or fragment includes a VH region consisting of the amino acid sequence of SEQ ID NO: 125. In one embodiment, the antibody or fragment includes a VL region containing the amino acid sequence of SEQ ID NO: 148 (1140_P01_G08)[G4_12]. In one embodiment, the antibody or fragment includes a VL region consisting of the amino acid sequence of SEQ ID NO: 148.

[0180] In one embodiment, the antibody or fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 125 and a VL region containing the amino acid sequence of SEQ ID NO: 148.

[0181] In one embodiment, the antibody or fragment thereof includes a VH region containing the amino acid sequence of SEQ ID NO: 119 (1139_P01_A04) [G4_3]. In one embodiment, the antibody The fragment contains a VH region consisting of the amino acid sequence of SEQ ID NO: 119. In one embodiment, the antibody or the fragment contains a VL region consisting of the amino acid sequence of SEQ ID NO: 142 (1139_P01_A04)[G4_3]. In one embodiment, the antibody or the fragment contains a VL region consisting of the amino acid sequence of SEQ ID NO: 142.

[0182] In one embodiment, the antibody or fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 119 and a VL region containing the amino acid sequence of SEQ ID NO: 142.

[0183] In one embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 129 (1248_P02_D10)[G4_16]. In one embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 129. In one embodiment, the antibody or fragment thereof comprises a VL region comprising the amino acid sequence of SEQ ID NO: 152 (1248_P02_D10)[G4_16]. In one embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 152.

[0184] In one embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 129 and a VL region comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 129 and a VL region consisting of the amino acid sequence of SEQ ID NO: 152.

[0185] In one embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 130 (1254_P01_H04)[G4_18]. In one embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 130. In one embodiment, the antibody or fragment thereof comprises a VL region comprising the amino acid sequence of SEQ ID NO: 153 (1254_P01_H04)[G4_18]. In one embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 153.

[0186] In one embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 130 and a VL region comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 130 and a VL region consisting of the amino acid sequence of SEQ ID NO: 153.

[0187] In one embodiment, the antibody or fragment includes a VH region containing the amino acid sequence of SEQ ID NO: 132 (1254_P02_G02)[G4_20]. In one embodiment, the antibody or fragment includes a VH region consisting of the amino acid sequence of SEQ ID NO: 132. In one embodiment, the antibody or fragment includes a VL region containing the amino acid sequence of SEQ ID NO: 155 (1254_P02_G02)[G4_20]. In one embodiment, the antibody or fragment includes a VL region consisting of the amino acid sequence of SEQ ID NO: 155.

[0188] In one embodiment, the antibody or fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 132 and a VL region containing the amino acid sequence of SEQ ID NO: 155.

[0189] In one embodiment, the antibody or fragment includes a VH region comprising the amino acid sequence of SEQ ID NO: 134 (1253_P03_H05)[G4_23]. In one embodiment, the antibody or fragment includes a VH region comprising the amino acid sequence of SEQ ID NO: 134. In one embodiment, the antibody or fragment includes a VH region comprising the amino acid sequence of SEQ ID NO: 157 (1253_P03_H05)[G4 It includes a VL region containing the amino acid sequence of [_23]. In one embodiment, the antibody or fragment thereof includes a VL region consisting of the amino acid sequence of SEQ ID NO: 157.

[0190] In one embodiment, the antibody or fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 134 and a VL region containing the amino acid sequence of SEQ ID NO: 157.

[0191] In one embodiment, the antibody or fragment includes a VH region containing the amino acid sequence of SEQ ID NO: 138 (1248_P02_C10)[G4_27]. In one embodiment, the antibody or fragment includes a VH region consisting of the amino acid sequence of SEQ ID NO: 138. In one embodiment, the antibody or fragment includes a VL region containing the amino acid sequence of SEQ ID NO: 161 (1248_P02_C10)[G4_27]. In one embodiment, the antibody or fragment includes a VL region consisting of the amino acid sequence of SEQ ID NO: 161.

[0192] In one embodiment, the antibody or fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 138 and a VL region containing the amino acid sequence of SEQ ID NO: 161.

[0193] In the case of a fragment containing both a VH region and a VL region, these can be linked either covalently (e.g., via a disulfide bond or linker) or non-covalently. The antibody fragments described herein may include scFv, i.e., fragments containing a VH region and a VL region linked by a linker. In one embodiment, the VH region and the VL region are linked by a (e.g., synthetic) polypeptide linker. The polypeptide linker is (Gly4Ser) n It may contain a linker, n=1 to 8, e.g., 2, 3, 4, 5, or 7. The polypeptide linker is [(Gly4Ser) n (Gly3AlaSer) m ] p The linker may include n=1 to 8, e.g., 2, 3, 4, 5, or 7, m=0 to 8, e.g., 0, 1, 2, or 3, and p=1 to 8, e.g., 1, 2, or 3. In a further embodiment, the linker includes sequence number 186. In a further embodiment, the linker consists of sequence number 186.

[0194] In one embodiment, the antibody or fragment comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 163-185. In a further embodiment, the antibody or fragment comprises an amino acid sequence of any one of SEQ ID NOs: 163-185. In yet another embodiment, the antibody or fragment comprises an amino acid sequence of SEQ ID NOs: 171, 165, 175, 176, 178, 180, or 184.

[0195] In one embodiment, the antibody or fragment comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 163 to 185. In a further embodiment, the antibody or fragment comprises an amino acid sequence with any one of SEQ ID NOs. 163 to 185. In yet another embodiment, the antibody or fragment comprises an amino acid sequence with SEQ ID NOs. 171, 165, 175, 176, 178, 180, or 184.

[0196] As described herein, the antibody may be of any form. In a preferred embodiment, the antibody is of type IgG1. Thus, in one embodiment, the antibody or a fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 233 to 255. In a further embodiment, the antibody or a fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs. 233 to 255. In yet another embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NOs. 235, 241, 245, 246, or 254.

[0197] In one embodiment, the antibody or fragment comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 233 to 255. In a further embodiment, the antibody or fragment comprises an amino acid sequence of any one of SEQ ID NOs. 233 to 255. In yet another embodiment, the antibody or fragment comprises an amino acid sequence of SEQ ID NOs. 235, 241, 245, 246, or 254.

[0198] Alternatively, an antibody or fragment thereof is provided, comprising a heavy chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 284 to 306 and / or a light chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 307 to 329. Thus, an antibody or fragment thereof is provided, comprising a heavy chain amino acid sequence according to any one of SEQ ID NOs. 284 to 306 and / or a light chain amino acid sequence according to any one of SEQ ID NOs. 307 to 329. In a particular embodiment, an antibody or fragment thereof is provided, comprising a heavy chain amino acid sequence according to SEQ ID NOs. 292 and / or a light chain amino acid sequence (clone G4_12) according to SEQ ID NOs. 315. In a further embodiment, an antibody or fragment thereof is provided, comprising a heavy chain amino acid sequence according to SEQ ID NOs. 286 and / or a light chain amino acid sequence (clone G4_3) according to SEQ ID NOs. 309. In a further embodiment, an antibody or fragment thereof is provided, comprising a heavy chain amino acid sequence according to SEQ ID NOs. 296 and / or a light chain amino acid sequence (clone G4_16) according to SEQ ID NOs. 319. In a further embodiment, an antibody or fragment thereof is provided, comprising or consisting of the heavy chain amino acid sequence according to SEQ ID NO: 297 and / or the light chain amino acid sequence (clone G4_18) according to SEQ ID NO: 320. In a further embodiment, an antibody or fragment thereof is provided, comprising or consisting of the heavy chain amino acid sequence according to SEQ ID NO: 299 and / or the light chain amino acid sequence (clone G4_20) according to SEQ ID NO: 322. In a further embodiment, an antibody or fragment thereof is provided, comprising or consisting of the heavy chain amino acid sequence according to SEQ ID NO: 301 and / or the light chain amino acid sequence (clone G4_23) according to SEQ ID NO: 324. In a further embodiment, an antibody or fragment thereof is provided, comprising or consisting of the heavy chain amino acid sequence according to SEQ ID NO: 305 and / or the light chain amino acid sequence (clone G4_27) according to SEQ ID NO: 328. In another embodiment, the antibody or fragment thereof is, (a) Heavy chain amino acid sequence according to SEQ ID NO: 284 and light chain amino acid sequence according to SEQ ID NO: 307 (clone G4_1), (b) Heavy chain amino acid sequence according to SEQ ID NO: 285 and light chain amino acid sequence according to SEQ ID NO: 308 (clone G4_2), (c) Heavy chain amino acid sequence according to SEQ ID NO: 287 and light chain amino acid sequence according to SEQ ID NO: 310 (clone G4_4), (d) Heavy chain amino acid sequence according to SEQ ID NO: 288 and light chain amino acid sequence according to SEQ ID NO: 311 (clone G4_5), (e) Heavy chain amino acid sequence according to SEQ ID NO: 289 and light chain amino acid sequence according to SEQ ID NO: 312 (clone G4_6), (f) Heavy chain amino acid sequence according to SEQ ID NO: 290 and light chain amino acid sequence according to SEQ ID NO: 313 (clone G4_7), (g) Heavy chain amino acid sequence according to SEQ ID NO: 291 and light chain amino acid sequence according to SEQ ID NO: 314 (clone G4_10), (h) Heavy chain amino acid sequence according to SEQ ID NO: 293 and light chain amino acid sequence according to SEQ ID NO: 316 (clone G4_13), (i) Heavy chain amino acid sequence according to SEQ ID NO: 294 and light chain amino acid sequence according to SEQ ID NO: 317 (clone G4_14), (j) Heavy chain amino acid sequence according to SEQ ID NO: 295 and light chain amino acid sequence according to SEQ ID NO: 318 (clone G4_15), (k) Heavy chain amino acid sequence according to SEQ ID NO: 298 and light chain amino acid sequence according to SEQ ID NO: 321 (clone G4_19), (l) Heavy chain amino acid sequence according to SEQ ID NO: 300 and light chain amino acid sequence according to SEQ ID NO: 323 (clone G4_22), (m) Heavy chain amino acid sequence according to SEQ ID NO: 302 and light chain amino acid sequence according to SEQ ID NO: 325 (clone G4_24), (n) Heavy chain amino acid sequence according to SEQ ID NO: 303 and light chain amino acid sequence according to SEQ ID NO: 326 (clone G4_25), (o) Heavy chain amino acid sequence according to SEQ ID NO: 304 and light chain amino acid sequence according to SEQ ID NO: 327 (clone G4_26), or, (p) Consists of or comprises the heavy chain amino acid sequence according to SEQ ID NO: 306 and the light chain amino acid sequence according to SEQ ID NO: 329 (clone G4_28).

[0199] Competitive antibody In one embodiment, an antibody or fragment thereof that specifically binds to the Vγ4 chain of the γδTCR but not to the Vγ2 chain of the γδTCR binds to, or competes with, the same or essentially the same epitope as, the antibody or fragment thereof defined or exemplified herein. By using routine methods known in the art, it is readily possible to determine whether an antibody binds to, or competes for, the same epitope as, the reference anti-Vγ4 antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-Vγ4 antibody described herein, the reference antibody can be conjugated to the Vγ4 protein or peptide under saturated conditions. The ability of the test antibody to bind to the Vγ4 chain is then evaluated. If the test antibody can bind to Vγ4 following saturated binding with the reference anti-Vγ4 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Vγ4 antibody. On the other hand, if the test antibody cannot bind to the Vγ4 chain after saturated binding with the reference anti-Vγ4 antibody, the test antibody may bind to the same epitope to which the reference anti-Vγ4 antibody binds.

[0200] This disclosure also includes anti-Vγ4 antibodies or fragments thereof that compete for binding to Vγ4 with antibodies having the CDR sequence of any of the antibodies or fragments thereof as defined herein, or with any of the exemplary antibodies described herein. For example, competitive assays can be performed using the antibodies described herein to determine which proteins, antibodies and other antagonists compete for binding to the Vγ4 chain with an antibody and / or share an epitope. These assays are readily known to those skilled in the art. Those skilled in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein, e.g., Vγ4. The antibody (or fragment thereof) is immobilized or insolubilized before and after competition, and the sample bound to the Vγ4 chain is separated from the unbound sample, for example, by decanting (if the antibody is insolubilized beforehand) or centrifugation (if the antibody precipitates after the competitive reaction). Competitive binding may also be determined by whether the function of the protein is altered by the binding or lack thereof of the antibody to the protein, for example, whether the antibody molecule inhibits or enhances the enzyme activity of, for example, a label. ELISA and other functional assays can be used as are known in the art and described herein.

[0201] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the target antigen. That is, one antibody in a 1x, 5x, 10x, 20x, or 100x or more excess inhibits the binding of the other by at least 50%, but preferably up to 75%, 90%, or 99%, as measured by a competitive binding assay. Alternatively, if essentially all amino acid mutations in the target antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope.

[0202] Next, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blockade (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0203] Modification of antibody sequences Antibodies and their fragments can be modified using known methods. Sequence modifications to antibody molecules described herein can be readily adopted by those skilled in the art. The following examples are not limiting.

[0204] During antibody discovery and sequence recovery from a phage library, the desired antibody variable domain can be reorganized into full-length IgG by subcloning. To accelerate the process, the variable domain is often transferred using restriction enzymes. These restriction sites can introduce additional / alternative amino acids and deviate from the canonical sequence (such canonical sequences can be found, for example, in the international ImMunoGeneTics [IMGT] information system; see http: / / www.imgt.org). These can be introduced as kappa or lambda light chain modifications.

[0205] Modification of Kappa Light Chain The variable sequences of kappa light chains can be cloned using a restriction site (e.g., Nhe1-Not1) when rearranged into full-length IgG. More specifically, an additional Ala-Ser sequence is introduced to the N-terminus of the kappa light chain to support cloning. Preferably, this additional AS sequence is subsequently removed during further development, such as to create a canonical N-terminal sequence. Thus, in one embodiment, kappa light chains containing the antibodies described herein do not contain an AS sequence at their N-terminus. That is, SEQ ID NOs. 140-147 and 156-158 do not contain the first AS sequence. It will be understood that this embodiment also applies to other sequences included herein that contain this sequence.

[0206] Additional amino acid modifications may be made to support cloning. For example, in the antibody described herein, a change from valine to alanine at the kappa light chain variable domain / constant domain boundary was introduced to support cloning when preparing the full-length sequence. This modified the kappa constant domain. Specifically, this modified the constant domain

[0207] [ka]

[0208] It will begin (from the NotI restriction site). Preferably, this sequence is

[0209] [ka]

[0210] The canonical kappa light chain constant region beginning with canonical kappa light chain may be modified during further development. Such modifications do not alter the functionality of the antibody. Therefore, in one embodiment, the kappa light chain containing the antibody described herein contains a constant domain beginning with sequence RTV. Therefore, in one embodiment, sequences 233-240, 249-251, 307-314 and 323-325

[0211] [ka]

[0212] is an array

[0213] [ka]

[0214] It can be replaced by. In a preferred embodiment including a preferred kappa light chain constant domain allotype, the kappa light chain constant domain has the amino acid sequence of SEQ ID NO: 330 and can be combined with any light chain variable domain disclosed herein.

[0215] Modification of Lambda Light Chain Similar to the kappa example described above, the variable domain of the lambda light chain can also be cloned by introducing a restriction site (e.g., Nhe1-Not1) during rearrangement into full-length IgG. More specifically, an additional Ala-Ser sequence may be introduced to the N-terminus of the lambda light chain to support cloning. Preferably, this additional AS sequence is subsequently removed during further development, such as to create a canonical N-terminal sequence. Thus, in one embodiment, the lambda light chains containing the antibodies described herein do not contain an AS sequence at their N-terminus; that is, SEQ ID NOs. 148-155 and 159-162 do not contain the first AS sequence. It will be understood that this embodiment also applies to other sequences included herein that contain this sequence.

[0216] As another example, in the case of the antibodies described herein, the change from lysine to alanine at the lambda light chain variable domain / constant domain boundary was introduced to support cloning when preparing the full-length sequence. This modified the lambda constant domain. Specifically, this resulted in the constant domain being

[0217] [ka]

[0218] It will begin (from the NotI restriction site). Preferably, this sequence is

[0219] [ka]

[0220] Further modifications may be made during development to create a canonical lambda light chain constant region beginning with GQPK. Such modifications do not alter the functionality of the antibody. Therefore, in one embodiment, the lambda light chain containing the antibody described herein contains a constant domain beginning with the sequence GQPK. Therefore, in one embodiment, sequences 241-248, 252-255, 315-322 and 326-329

[0221] [ka]

[0222] is an array

[0223] [ka]

[0224] It can be replaced by. In a preferred embodiment that includes a preferred lambda light chain constant domain allotype, the lambda light chain constant domain has the amino acid sequence of SEQ ID NO: 331 and can be combined with any light chain variable domain disclosed herein.

[0225] Modification of Lambda Light Chain and Kappa Light Chain In light of the above disclosure relating to the removal of N-terminal AS residues from lambda and / or kappa light chain variable domains disclosed herein as SEQ ID NOs: 140-162, isolated anti-Vγ4 antibodies or fragments may contain light chain variable (VL) amino acid sequences by any one of SEQ ID NOs: 261-283, corresponding to SEQ ID NOs: 140-162 lacking N-terminal AS residues. Accordingly, any reference herein to VL amino acid sequences by one or more SEQ ID NOs: 140-162 can be replaced by VL amino acid sequences by SEQ ID NOs: 261-283, respectively, and all such embodiments are disclosed herein. For example, a reference herein to a light chain variable domain by SEQ ID NO: 148 (derived from clone G4_12) can be replaced with a reference to SEQ ID NO: 269.

[0226] Modification of heavy chain Typically, human variable heavy chain sequences begin with either basic glutamine (Q) or acidic glutamic acid (E). However, both such sequences are known to be converted to pyroglutamic acid (pE), an acidic amino acid residue. The conversion from Q to pE alters the antibody's charge, while the conversion from E to pE does not. Therefore, one option to avoid fluctuations in charge change over time is to initially modify the starting heavy chain sequence from Q to E. Thus, in one embodiment, the heavy chain of the antibody described herein having a Q residue at its N-terminus may contain a Q-to-E modification at the N-terminus. In particular, the first residue of any of sequence numbers 118, 120, 124, 126, 132, 133, 135, 137, 138 and / or 139 may be modified from Q to E. It will be understood that this embodiment also applies to other sequences included herein that contain this sequence (i.e., any embodiment incorporating these sequences, for example, into a full-length antibody or a fragment thereof). In some embodiments, it may be advantageous to substitute the N-terminal E residue of the heavy chain with a Q residue. Thus, in some embodiments, the N-terminal E residue of any one of sequence numbers 117, 119, 121-123, 125, 127-131, 134 and / or 136 may be substituted with a Q residue.

[0227] Furthermore, the C-terminus of the IgG1 constant domain terminates with PGK. However, the further terminal basic lysine (K) is often cleaved during expression (e.g., in CHO cells). This alters the antibody's charge due to the various loss of C-terminal lysine residues. Therefore, one option is to remove the lysine first to obtain a uniform and consistent heavy-chain C-terminal sequence terminated with PG. Thus, in one embodiment, the heavy chain of the antibody described herein has a terminal K removed from its C-terminus. In particular, the antibody may include one of SEQ ID NOs. 233-255 or 284-306, in which the terminal lysine residue has been removed.

[0228] Modification of any Arotype During antibody discovery, a specific human allotype may be used. Optionally, antibodies can be switched to different human allotypes during development. As a non-limiting example, the kappa chain has three human allotypes called Km1, Km1,2, and Km3, which define three Km alleles (using allotype numbering). Km1 correlates with valine 153 (IMGT V45.1) and leucine 191 (IMGT L101), Km1,2 correlates with alanine 153 (IMGT A45.1) and leucine 191 (IMGT L101), and Km3 correlates with alanine 153 (IMGT A45.1) and valine 191 (IMGT V101) ) correlates with ). Therefore, by arbitrary selection, the sequence can be modified from one allotype to another by a standard cloning approach. For example, modifying L191V (IMGT L101V) converts the Km1,2 allotype to the Km3 allotype. For details on such allotypes, see Jefferis and Lefranc (2009) MAbs1(4):332-8, which is incorporated herein by reference.

[0229] Thus, in one embodiment, the antibodies described herein contain amino acid substitutions derived from another human allotype of the same gene. In a further embodiment, the antibody contains an L191V (IMGT L101V) substitution in the kappa chain for converting the c domain from km1,2 to the km3 allotype.

[0230] In a preferred embodiment including a preferred kappa light chain constant domain allotype, the kappa light chain constant domain has the amino acid sequence according to SEQ ID NO: 330 and can be combined with any light chain variable domain disclosed herein. In an alternative preferred embodiment including a preferred lambda light chain constant domain allotype, the lambda light chain constant domain has the amino acid sequence according to SEQ ID NO: 331 and can be combined with any light chain variable domain disclosed herein.

[0231] Antibody binding The antibody or fragment thereof can bind to the Vγ4 chain of γδTCR with a binding affinity (KD) less than 3.0×10 -7 M (i.e., 300 nM) or less than 1.5×10 -7 M (i.e., 150 nM). In a further embodiment, the KD is 1.3×10 -7 M (i.e., 130 nM) or less, such as 1.0×10 -7 M (i.e., 100 nM) or less. In yet another embodiment, the KD is less than 6.0×10 -8 M (i.e., 60 nM), such as less than 5.0×10 -8 M (i.e., 50 nM), less than 4.0×10 -8 M (i.e., 40 nM), less than 3.0×10 -8 M (i.e., 30 nM) or less than 2.0×10 -8 M (i.e., 20 nM). In a further embodiment, the KD is 1.0×10 -8 M (i.e., 10 nM) or less, such as 5.0×10 -9 M (i.e., 5 nM) or less, 4.0×10 -9 M (i.e., 4 nM) or less, 3.0×10 -9M (i.e., 3nM) or less, 2.0 × 10 -9 M (i.e., 2nM) or less or 1.0 × 10 -9 It may be less than or equal to M (i.e., 1 nM). For example, in one embodiment, the (e.g., human) anti-Vγ4 antibody is measured by surface plasmon resonance and is 1.5 × 10⁻⁶ -7 It binds to the Vγ4 chain of the γδTCR with a binding affinity (KD) of less than M (i.e., 150 nM).

[0232] In one embodiment, the antibody or a fragment thereof is measured by surface plasmon resonance at 4.0 × 10⁶ -8 Less than M (i.e., 40 nM), 3.0 × 10 -8 Less than M (i.e., 30 nM) or 2.0 × 10 -8 It binds to the Vγ4 chain of the γδTCR with a binding affinity (KD) of less than M (i.e., 20 nM).

[0233] In one embodiment, the binding affinity of an antibody or fragment is established by directly or indirectly coating the surface of a sensor (e.g., an amine high-capacity chip or equivalent) with the antibody or fragment (e.g., by capture with anti-human IgG Fc), and the target (i.e., the Vγ4 chain of the γδTCR) bound by the antibody or fragment flows over the chip to detect binding. In an alternative embodiment, an antigen may be coated directly or indirectly onto the surface of the sensor over which the test antibody or fragment flows. Those skilled in the art can easily determine appropriate test conditions. For example, a MASS-2 instrument (also known as Sierra SPR-32) can be appropriately used at 25°C at a rate of 30 μl / min in PBS + 0.02% Tween 20 electrophoresis buffer. In a suitable embodiment, the Reichert4SPR instrument can be used at room temperature (e.g., 25°C) with a flow rate of 25 μl / min in PBS + 0.05% Tween20.

[0234] Functional properties of antibodies Assays that may be used to define antibody function are described herein. For example, antibodies or fragments thereof described herein may be evaluated by measuring the involvement of γδTCR, e.g., downregulation of γδTCR and / or upregulation of CD69 surface expression upon antibody binding. Surface expression of γδTCR or CD69 after application of the antibody or fragment thereof (optionally presented on the surface of cells) can be measured, for example, by flow cytometry.

[0235] The antibodies or fragments described herein can also be evaluated by measuring γδT cell degranulation. For example, the expression of CD107a, a marker of cell degranulation, after application of the antibody or fragment to γδT cells (optionally presented on the cell surface) can be measured, for example, by flow cytometry. The antibodies or fragments described herein can also be evaluated by measuring γδT cell-mediated death activity (to test whether the antibody affects the death activity of γδT cells, i.e., the ability of the antibody to induce γδT cells to kill target cells directly or indirectly). For example, target cells can be incubated with γδT cells in the presence of the antibody or fragment (optionally presented on the cell surface). After incubation, the culture can be stained with a cell viability dye to distinguish between living and dead target cells. The percentage of dead cells can then be measured, for example, by flow cytometry.

[0236] As described herein, antibodies or fragments used in assays may be presented on a surface, such as the surface of a cell containing an Fc receptor. For example, an antibody or fragment may be presented on the surface of THP-1 cells, such as TIB-202® cells (available from the American Type Culture Collection (ATCC)). Alternatively, the antibody or fragment may be used directly in the assay.

[0237] In such functional assays, the output can be measured by calculating half of the maximum concentration, also referred to as "EC50" or "50% effective concentration." The term "IC50" refers to the inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, such as flow cytometry. To avoid misunderstanding, the EC50 values ​​in this application are provided using IgG1 antibodies. Such values ​​can be easily converted to equivalent values ​​based on the molecular weight of the antibody form, as follows: (μg / ml) / (MW in kDa) = μM

[0238] In this specification, milliliters are written as "ml" or "mL" and can be used interchangeably.

[0239] The EC50 for downregulating γδTCR upon antibody (or fragment) binding may be less than 0.5 μg / ml, for example, less than 0.4 μg / ml, 0.3 μg / ml, 0.2 μg / ml, 0.15 μg / ml, 0.1 μg / ml, or 0.05 μg / ml. In particular, the EC50 values ​​are those measured when the antibody is of IgG1 type. For example, the EC50 γδTCR downregulation value can be measured using flow cytometry.

[0240] The EC50 of γδ T cell degranulation upon antibody (or fragment) binding may be less than 0.05 μg / ml, for example, less than 0.04 μg / ml, 0.03 μg / ml, 0.02 μg / ml, 0.015 μg / ml, 0.01 μg / ml, or 0.008 μg / ml. In particular, the EC50 values ​​are those measured when the antibody is of type IgG1. For example, the EC50 value of γδ T cell degranulation can be measured using flow cytometry to assess CD107a expression (i.e., cell CD107a expression can be measured by detecting a degranulation marker. In one embodiment, CD107a expression is measured using an anti-CD107a antibody such as anti-human CD107aBV421 (clone H4A3) (BD Biosciences).

[0241] The EC50 of γδT cell-mediated cell death upon antibody (or fragment) binding may be less than 0.5 μg / ml, for example, less than 0.4 μg / ml, 0.3 μg / ml, 0.2 μg / ml, 0.15 μg / ml, 0.1 μg / ml, or 0.07 μg / ml. In particular, the EC50 values ​​are those measured when the antibody is of IgG1 type. For example, the EC50 γδT cell-mediated cell death value can be measured by detecting the percentage of dead cells using flow cytometry after incubation of the antibody, γδT cells, and target cells (i.e., using a cell viability dye). In one embodiment, target cell death is measured using the cell viability dye Viability Dye eFluor® 520 (ThermoFisher).

[0242] In the assays described in these embodiments, the antibody or a fragment thereof may be presented on the surface of cells such as THP-1 cells, for example, TIB-202® (ATCC). The THP-1 cells are optionally labeled with a dye such as CellTracker® Orange CMTMR (ThermoFisher).

[0243] Polynucleotides and expression vectors Polynucleotides encoding anti-Vγ4 antibodies or fragments described herein are also provided. In one embodiment, the anti-Vγ4 antibody or fragment is encoded by a polynucleotide containing, or comprising, sequences having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99% sequence identity with SEQ ID NOs. 187-232. In one embodiment, the anti-Vγ4 antibody or fragment is encoded by an expression vector containing the VH region of SEQ ID NOs. 187-209. In another embodiment, the anti-Vγ4 antibody or fragment is encoded by an expression vector containing the VL region of SEQ ID NOs. 210-232. In a further embodiment, the anti-Vγ4 antibody or fragment is encoded by a polynucleotide containing, or comprising, SEQ ID NOs. 187-232. In a further embodiment, a cDNA containing the polynucleotide is provided.

[0244] In one embodiment, the polynucleotide includes or comprises a sequence having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, with sequence identity with SEQ ID NOs: 195, 189, 199, 200, 202, 204, 208, 218, 212, 222, 223, 225, 227, or 231. In one embodiment, the expression vector includes the VH region of SEQ ID NOs: 195, 189, 199, 200, 202, 204, or 208. In another embodiment, the expression vector includes the VL region of SEQ ID NOs: 218, 212, 222, 223, 225, 227, or 231. In a further embodiment, the polynucleotides include, or consist of, SEQ ID NOs: 195, 189, 199, 200, 202, 204, 208, 218, 212, 222, 223, 225, 227, or 231, particularly SEQ ID NOs: 195 and / or 218, or SEQ ID NOs: 189 and / or 212. In a further embodiment, a cDNA comprising the polynucleotides is provided.

[0245] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%, sequence identity with any one of the portions of SEQ ID NOs. 187-232 encoding CDR1, CDR2, and / or CDR3 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises the encoded immunoglobulin chain The sequence comprises or consists of sequences having at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99%, e.g., at least 99%, e.g., sequence 195, 189, 199, 200, 202, 204, 208, 218, 212, 222, 223, 225, 227, or 231 sequence codes encoding CDR1, CDR2, and / or CDR3 of the robulin chain variable domain.

[0246] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, with any one of the portions of SEQ ID NOs: 187-232 encoding FR1, FR2, FR3 and / or FR4 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, with SEQ ID NOs: 195, 189, 199, 200, 202, 204, 208, 218, 212, 222, 223, 225, 227, or 231 encoding FR1, FR2, FR3 and / or FR4 of the encoded immunoglobulin chain variable domain.

[0247] To express an antibody or a fragment thereof, polynucleotides encoding a light chain and a heavy chain of partial or full length as described herein are inserted into an expression vector such that the gene is operably bound to a transcriptional and translational control sequence (which may be referred to as an “expression cassette” as is well understood in the art). Thus, expression vectors comprising the polynucleotide sequences as defined herein are also described. In one embodiment, the expression vector comprises a VH sequence of any one of SEQ ID NOs: 187-209, such as any one of SEQ ID NOs: 195, 189, 199, 200, 202, 204, or 208. In another embodiment, the expression vector comprises a VL region of any one of SEQ ID NOs: 210-232, such as any one of SEQ ID NOs: 218, 212, 222, 223, 225, 227, or 231. Such expression vectors or cassettes may be used in pairs, with the heavy chain variable sequence and the light chain variable sequence appropriately paired according to the pairing of various amino acid sequences that provide the antibodies disclosed herein. In some embodiments, the expression vector comprises a sequence having at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99% or 100% sequence identity with any one of SEQ ID NOs: 187-209 (encoding variable weight regions), and further comprises a sequence having at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g., at least 99% or 100% sequence identity with any one of SEQ ID NOs: 210-232 (encoding variable light regions). Here again, the sequences may be provided in specific pairs as described herein to encode the antibodies described herein.

[0248] Polynucleotides and expression vectors may also be described by reference to the encoded amino acid sequence. Thus, in one embodiment, a polynucleotide comprises or consists of a sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1-186, 233-260.

[0249] Mutations can be induced in the DNA or cDNA encoding polypeptides that are silent in terms of the polypeptide's amino acid sequence but provide preferred codons for translation in a particular host. For example, preferred codons for nucleic acid translation are known in E. coli and S. cerevisiae, as well as in mammals, particularly humans.

[0250] Polypeptide mutations can be achieved, for example, by substitution, addition, or deletion of the nucleic acid encoding the polypeptide. Substitution, addition, or deletion of the nucleic acid encoding the polypeptide can be achieved, for example, by error-prone PCR, mixed, or oligonucleotide-specific mutation. Nucleic acid modifications, additions, or deletions can also be introduced by methods including assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recurrent ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene rearrangement, artificial gene synthesis, site-saturated mutagenesis (GSSM), synthetic ligation rearrangement (SLR), or combinations thereof.

[0251] In particular, artificial gene synthesis can be used. Genes encoding polypeptides can be synthesized synthetically, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo without the need for a precursor template DNA. To obtain the desired oligonucleotide, the components are sequentially attached to a growing oligonucleotide chain in the order required by the product sequence. Once chain construction is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity.

[0252] Expression vectors include, for example, plasmids, retroviruses, cosmids, yeast artificial chromosomes (YACs), and Epstein-Barr virus (EBV)-derived episomes. Polynucleotides are ligated into the vector so that transcription and translation control sequences within the vector perform their intended functions of regulating the transcription and translation of the polynucleotides. Expression and / or control sequences may include promoters, enhancers, transcription terminators, the 5' start codon (i.e., ATG) of the coding sequence, intron splicing signals, and stop codons. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. Accordingly, nucleotide sequences encoding the single-stranded variable fragment of the present invention by any one of SEQ ID NOs. 163-185, including VH and VL regions linked by a synthetic linker (encoding SEQ ID NO: 186), are also described. It will be understood that the polynucleotides or expression vectors described herein may include the VH region, the VL region, or both (including the linker, optionally). Therefore, polynucleotides encoding the VH and VL regions can be inserted into separate vectors, or sequences encoding both regions can be inserted into the same expression vector. The polynucleotide(s) are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites in the polynucleotide and vector, or blunt-end ligation if no restriction enzyme sites are present).

[0253] A convenient vector encodes a functionally complete human CH or CL immunoglobulin sequence and features appropriate restriction sites engineered to facilitate the insertion and expression of any VH or VL sequence, as described herein. Expression vectors can also encode signal peptides that promote the secretion of antibodies (or fragments thereof) from host cells. Polynucleotides can be cloned into vectors such that the signal peptide is conjugated in-frame to the amino terminus of the antibody. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0254] The host cell may contain a first vector encoding the light chain of the antibody or a fragment thereof, and a second vector encoding the heavy chain of the antibody or a fragment thereof. Alternatively, both the heavy and light chains may be encoded on the same expression vector introduced into the host cell.

[0255] In one embodiment, a polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or fragment thereof, and the antibody or fragment thereof is presented on the extracellular surface of a host cell.

[0256] Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, transduction, encapsulation of polynucleotides into liposomes, gene gun injection, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0257] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other usable cell lines are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Antigen-binding fragments of antibodies, such as scFv and Fv fragments, can be isolated and expressed in E. coli using methods known in the art.

[0258] Antibodies are produced by culturing host cells for a period of time sufficient to allow antibody expression in the host cells, or more preferably, the secretion of antibodies into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0259] The antibodies (or fragments) described herein can be obtained and manipulated using, for example, the techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th Edition, Cold Spring Harbour Laboratory Press.

[0260] Monoclonal antibodies can be produced by using hybridoma technology to fuse specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to proliferate in tissue culture and their lack of antibody chain synthesis.

[0261] Monoclonal antibodies against the determined antigen are, for example, a) Immortalizing lymphocytes obtained from the peripheral blood of animals previously immunized with immortal cells, and preferably myeloma cells, with the determined antigen in order to form hybridomas, b) This can be obtained by culturing the formed immortalized cells (hybridomas) and recovering cells that produce antibodies with the desired specificity.

[0262] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the target antigens described herein can be isolated from a suitable antibody library through routine procedures using, for example, phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Therefore, monoclonal antibodies can be, for example, a) Cloning the vector to DNA or cDNA sequences (appropriately immunized with determined antigens) obtained from phages and more specifically filamentous bacteriophages, lymphocytes, and especially peripheral blood lymphocytes of animals, b) Transforming prokaryotic cells with the above vector under conditions that enable antibody production, c) Selecting antibodies by subjecting them to antigen affinity selection, d) The antibody having the desired specificity can be obtained by a process that includes the steps of recovering the antibody.

[0263] Pharmaceutical composition A further aspect of the present invention provides a composition comprising a population of Vγ4T cells obtained by a method defined herein. In one embodiment, the Vγ4T cell population is a proliferated population of Vγ4T cells. In such embodiments, the composition may optionally contain cells in combination with other excipients. The composition also includes one or more additional activators (e.g., activators suitable for treating the diseases described herein).

[0264] Pharmaceutical compositions may comprise Vγ4T cells, particularly proliferated Vγ4T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used with the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration.

[0265] In one embodiment, the pharmaceutical composition is substantially free of contaminants such as endotoxins or mycoplasmas, for example, the contaminants are not present at detectable levels.

[0266] The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal). In some embodiments, the composition is administered by intravenous infusion or injection. In another preferred embodiment, the composition is administered by intramuscular or subcutaneous injection.

[0267] It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic methods for the treatment of the diseases described herein, either as an adjunct to or in combination with other established treatments commonly used for the treatment of such diseases.

[0268] In a further embodiment of the present invention, a cell population, composition, or pharmaceutical composition is administered sequentially, simultaneously, or separately, together with at least one activator.

[0269] Therapeutic methods using cell populations Further aspects of the present invention provide a cell population obtained by a method defined herein for use as a pharmaceutical agent. Further aspects of the present invention provide a proliferated cell population defined herein for use as a pharmaceutical agent. References herein to cell populations "for use" as a pharmaceutical agent or in therapy are limited to the administration of the cell population to a subject. Such use does not include the direct administration of an antibody or fragment thereof to a patient, i.e., when the antibody is used as a therapeutic agent.

[0270] In one embodiment, the cell population is for use in the treatment of cancer, infectious diseases, or inflammatory diseases. In a further embodiment, the cell population is for use in the treatment of cancer. That is the case.

[0271] In one embodiment, the cell population for use as a drug comprises more than 50% Vγ4T cells, for example, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% Vγ4T cells. In a further embodiment, the cell population for use as a drug comprises Vγ4T cells.

[0272] A further embodiment of the present invention provides a pharmaceutical composition comprising a population of cells as defined herein for use as a drug. In one embodiment, the pharmaceutical composition comprising the population of cells is for therapeutic use, and in particular for the treatment of cancer, infectious diseases or inflammatory diseases. In a further embodiment, the pharmaceutical composition comprising the population of cells is for the treatment of cancer.

[0273] A further aspect of the present invention provides a method for modulating the immune response of a target requiring such modification, comprising administering a therapeutically effective amount of a population of cells as defined herein.

[0274] A further aspect of the present invention provides a method for treating a target cancer, infection, or inflammatory disease requiring such treatment, comprising administering a therapeutically effective amount of a cell population as defined herein, or administering a pharmaceutical composition containing a therapeutically effective amount of a cell population.

[0275] A further aspect of the present invention provides the use of a cell population as defined herein for the manufacture of a drug, for example, in the treatment of cancer, infectious diseases, or inflammatory diseases.

[0276] Adoptive T cell therapy Gamma delta T cells obtained by the proliferation method of the present invention can be used, for example, as agents for adoptive T cell therapy. This includes transplantation of γδ T cells into a patient. The therapy may be autologous transplantation, i.e., γδ T cells may be transplanted again into the same patient from whom they were obtained, or the therapy may be allogeneic, i.e., γδ T cells from one person may be transplanted into another patient. If allogeneic transplantation is involved, the γδ T cells may not substantially contain αβ T cells. For example, αβ T cells may be depleted from the γδ T cell population, for example, after proliferation, using any suitable means known in the art (e.g., by negative selection using magnetic beads). The therapeutic method includes providing a sample obtained from a donor individual (e.g., a non-hematopoietic tissue sample), culturing the γδ T cells obtained from the sample as described herein to produce, for example, a proliferation population, and administering the population of γδ T cells to a recipient individual.

[0277] The patients or subjects to be treated are preferably human cancer patients (e.g., human cancer patients receiving treatment for solid tumors) or patients with viral infections (e.g., CMV-infected patients or HIV-infected patients). In some cases, patients are receiving treatment for solid tumors and / or are undergoing treatment for solid tumors. Because they are typically present in non-hematopoietic tissues, Vγ4 T cells present in tissues are also more likely to return to and be retained in tumor masses than their counterparts present in the systemic blood, and adoptive transplantation of these cells is likely to be more effective when targeting solid tumors and potentially other non-hematopoietic tissue-associated immunopathologies.

[0278] Because γδ T cells are non-MHC restricted, they do not recognize the recipient host as foreign, meaning they are less likely to cause graft-versus-host disease. This means they can be used "off-the-shelf" and transplanted into any recipient, for example, for allogeneic adoptive T cell therapy.

[0279] The γδT cells obtained by the method described herein express NKG2D and are used in malignant tumors. They respond to NKG2D ligands (e.g., MICA) that are strongly associated with this. They can also express cytotoxic profiles even in an inactive state and may therefore be effective in killing tumor cells. For example, γδT cells obtained as described herein may express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulisin, granzyme A and B, and perforin even in an inactive state. IL-17A may not be expressed.

[0280] In some embodiments, a method for treating an individual with a tumor may include providing a sample of the tumor obtained from a donor individual, culturing γδT cells obtained from the sample as described above, and administering the population of γδT cells to the individual with the tumor. In further embodiments, a method for treating an individual with a tumor in non-hematopoietic tissue may include providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδT cells obtained from the sample as described above, and administering the population of γδT cells to the individual with the tumor.

[0281] In some cases, a therapeutically effective amount of γδT cells obtained by any of the above methods can be administered to the subject in a therapeutically effective dose (e.g., for the treatment of cancer, e.g., for the treatment of solid tumors). In some cases, a therapeutically effective amount of γδT cells (e.g., Vγ4T cells) can be 10x10 12 Cells / dose less than (e.g., 9x10) 12 Cells / dose less than 8x1012 cells / dose, 7x10 12 Cells / dose less than 6x10 12 Cells / dose less than 5x10 12 Cells / dose less than 4x10 12 Cells / dose less than 3x10 12 Cells / dose less than 2x10 12 Cells / dose less than 1x10 12 Cells / dose less than 9x10 11 Cells / dose less than 8x10 11 Cells / dose less than 7x10 11 Cells / dose less than 6x10 11 Cells / dose less than 5x10 11 Cells / dose less than 4x10 11 Cells / dose less than 3x10 11 Cells / dose less than 2x10 11 Cells / dose less than 1x10 11 Cells / dose less than 9x10 10 Cells / dose less than 7.5 x 10 10 Cells / dose less than 5x10 10 Cells / dose less than 2.5 x 10 10 Cells / dose less than 1x10 10 Cells / dose less than 7.5 x 10 9 Cells / dose less than 5x10 9 Cells / dose less than 2.5 x 10 9 Cells / dose less than 1x10 9 Cells / dose less than 7.5 x 10 8 Cells / dose less than 5x10 8 Cells / dose less than 2.5 x 10 8 Cells / dose less than 1x10 8 Cells / dose less than 7.5 x 10 7 Cells / dose less than 5x10 7 Cells / dose less than 2.5 x 10 7 Cells / dose less than 1x10 7 Cells / dose less than 7.5 x 10 6 Cells / dose less than 5x10 6 Cells / dose less than 2.5 x 10 6 Cells / dose less than 1x10 6 Cells / dose less than 7.5 x 10 5 Cells / dose less than 5x105 Cells / less than the dosage, 2.5x10 5 Cells / less than the dosage, or 1x10 5 Cells / less than the dosage).

[0282] In some embodiments, a therapeutically effective amount of γδ T cells (e.g., Vγ4 T cells) is less than 10x10 12 Cells (e.g., during the treatment period, less than 9x10 12 Cells, less than 8x10 12 Cells, less than 7x10 12 Cells, less than 6x10 12 Cells, less than 5×10 12 Cells, less than 4×10 12 Cells, less than 3×10 12 Cells, less than 2×10 12 Cells, less than 1×10 12 Cells, less than 9×10 11 Cells, less than 8x10 11 Cells, less than 7x10 11 Cells, less than 6x10 11 Cells, less than 5x10 11 Cells, less than 4x10 11 Cells, less than 3x10 11 Cells, less than 2×10 11 Cells, less than 1×10 11 Cells, less than 9×10 10 Cells, less than 7.5×10 10 Cells, less than 5×10 10 Cells, less than 2.5×10 10 Cells, less than 1x10 10 Cells, less than 7.5x10 9 Cells, less than 5x10 9 Cells, less than 2.5x10 9 Cells, less than 1x10 9 Cells, less than 7.5x10 8 Cells, less than 5x10 8 Cells, less than 2.5×10 8 Cells, less than 1×10 8 Cells, less than 7.5×10 7 Cells, less than 5×10 7 Cells, less than 2.5×10 7 Cells, less than 1x10 7 Cells, less than 7.5x10 6Subcellular, 5x10 6 Subcellular, 2.5 x 10 6 Less than a cell, 1 x 10 6 Subcellular, 7.5 x 10 5 Subcellular, 5x10 5 Subcellular, 2.5 x 10 5 Less than a cell, or 1x10 5 It is less than a cell.

[0283] In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) described herein is approximately 1 x 10⁻¹⁶ 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , or 5x10 8 Contains cells / kg. In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) described herein is up to approximately 1 x 10⁻⁶. 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , or 5x10 8 Contains cells / kg. In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) is approximately 1.1 x 10⁻⁶. 6 ~1.8x10 7 Contains cells / kg. In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) is approximately 1 x 10⁻⁶. 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8, 1x10 9 , 2x10 9 , or 5x10 9 The cells are included. In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) is at least about 1 x 10⁻¹⁶ 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 Contains cells. In some embodiments, the dose of γδT cells (e.g., Vγ4T cells) is up to approximately 1 x 10⁻¹⁶ 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 Contains cells.

[0284] In one embodiment, the target is 10 per 1 kg of the target's body weight. 4 ~10 6 The subject is administered γδT cells (e.g., Vγ4T cells). In one embodiment, the subject receives an initial dose of the population of γδT cells (e.g., 10 per kg of the subject's body weight). 4 ~10 6 For example, 10 γδT cells per kg of the subject's body weight. 4 ~10 5 (10 gamma-δ T cells), followed by one or more (e.g., 2, 3, 4, or 5) doses of gamma-δ T cells (e.g., 10 per kg of the subject's body weight). 4 ~10 6 For example, 10 γδT cells per kg of the subject's body weight. 4 ~10 5The subject receives (10 γδT cells). In one embodiment, one or more subsequent doses are administered less than 15 days after the previous dose, for example, less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous dose, for example, less than 4, 3, or 2 days after the previous dose. In one embodiment, the subject receives a total of approximately 10 γδT cells per kg of body weight during the period of at least three doses of the γδT cell population. 6 Receiving individual γδT cells, for example, the subject is 1 x 10 5 Initial administration of γδT cells, 3 x 10 5 Second administration of γδT cells and 6x10 5 The third dose of γδT cells is administered, for example, less than 4, 3, or 2 days after the previous dose.

[0285] In some embodiments, one or more additional therapeutic agents may be administered to the subject. These additional therapeutic agents may be selected from a group consisting of immunotherapeutic agents, cytotoxic agents, proliferation inhibitors, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof. The additional therapeutic agents may be administered concurrently with, before, or after the administration of γδT cells. The additional therapeutic agents may be immunotherapeutic agents capable of acting on targets within the subject's body (e.g., the subject's own immune system) and / or on transplanted γδT cells.

[0286] The compositions can be administered by any convenient method. The compositions described herein may be administered to patients by intra-arterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular intravenous injection, or intraperitoneal injection, for example, by intradermal or subcutaneous injection. The γδT cell compositions can be injected directly into tumors, lymph nodes, or infected sites.

[0287] Genetic manipulation The γδ T cells obtained by the method of the present invention can also be genetically engineered to enhance their therapeutic properties for use in chimeric antigen receptor T cell (CAR-T) therapy and other applications. This involves generating engineered T cell receptors (TCRs) and reprogramming T cells with new specificity (e.g., specificity to monoclonal antibodies). Engineered TCRs can make T cells specific to malignant cells and therefore may be useful in cancer immunotherapy. For example, T cells may be used in target tissue or These CAR-modified T cells can recognize cancer cells that express tumor antigens, such as tumor-associated antigens, which are not expressed by normal somatic cells. Therefore, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0288] Other uses of antibodies or fragments thereof According to a further aspect of the present invention, the use of anti-Vγ4 antibodies or fragments thereof described herein is provided for studying antigen recognition, activation, signal transduction, or function of γδT cells (particularly Vγ4T cells). As described herein, the antibodies have been shown to be active in assays that can be used to investigate γδT cell function. Such antibodies may also be useful in inducing the proliferation of γδT cells and can therefore be used in methods for proliferating γδT cells (such as Vγ4T cells).

[0289] Antibodies that bind to the Vγ4 chain can be used to detect γδT cells. For example, the antibody may be labeled with a detectable label or reporter molecule, or it may be used as a capture ligand for selectively detecting and / or separating Vγ4T cells in a sample. Labeled antibodies have been used in many methods known in the art, such as immunohistochemistry and ELISA.

[0290] Detectable labels or reporter molecules are: 3 H, 14 C, 32 P, 35 S or 125The fluorescent label may be a radioactive isotope such as l, a fluorescent or chemiluminescent site such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Next, the fluorescent label applied to the antibody of the present invention can be used in fluorescent cell sorting (FACS).

[0291] It will be understood that all embodiments described herein are applicable to all aspects of the present invention.

[0292] Clause The following is a set of clauses defining the present invention and its preferred embodiments.

[0293] 1. An ex vivo method for modulating gamma variable 4 (Vγ4) T cells, comprising administering an antibody or fragment thereof that specifically binds to the Vγ4 chain of the γδ T cell receptor (TCR) but does not bind to the gamma variable 2 (Vγ2) chain of the γδ TCR to a cell population containing Vγ4 T cells.

[0294] 2. The method as defined in Clause 1, wherein the Vγ4 chain of the γδTCR is human Vγ4 and the Vγ2 chain of the γδTCR is human Vγ2.

[0295] 3. The method as defined in clause 1 or 2, wherein the antibody or fragment thereof binds to an epitope of the Vγ4 chain of the γδTCR, which includes one or more amino acid residues within the amino acid region 67-82 of SEQ ID NO: 1.

[0296] 4. The method as defined in any one of the clauses 1 to 3, wherein the antibody or fragment thereof binds to an epitope of the Vγ4 chain of the γδTCR, which includes one or more amino acid residues within the amino acid region 71 to 79 of SEQ ID NO: 1.

[0297] 5. The method as defined in Clause 4, wherein the epitope includes at least one of the amino acid residues 71, 73, 75, 76, and 79 of Sequence ID No. 1.

[0298] 6. The epitope consists of one or more amino acid residues within the amino acid region 67-82 of Sequence ID No. 1, as defined in any one of clauses 1-5.

[0299] 7. The epitope is defined in any one of the clauses 1 to 6, including or consisting of K76 and / or M80 of Sequence ID No. 1.

[0300] 8. The method defined in any one of clauses 1 to 7, wherein the epitope is an activating epitope of γδT cells.

[0301] 9. The binding of the activated epitope is the method defined in Clause 8, which (i) downregulates the γδTCR, (ii) activates the degranulation of the γδT cells, (iii) activates γδT cell-mediated death, and / or (iv) activates or increases cell signaling via the Vγ4 chain.

[0302] 10. A method defined in any one of clauses 1 to 9, wherein the antibody or fragment thereof binds only to the epitope in the V region of the Vγ4 chain of the γδTCR.

[0303] 11. The antibody or fragment thereof does not bind to the epitope found in CDR3 of the Vγ4 chain of the γδTCR, as defined in any one of the clauses 1 to 10.

[0304] 12. A CDR3 containing one of sequence numbers 2 to 47, preferably a sequence having at least 80% sequence identity with sequence number 10 and / or sequence number 33. CDR2 includes one of sequence numbers 48-70 and sequences A1-A23 (in Figure 1), preferably sequence number 56 and / or sequence A9, and / or a sequence having at least 80% sequence identity. An anti-Vγ4 antibody or a fragment thereof, comprising one or more CDR1 sequences having at least 80% sequence identity with any one of SEQ ID NOs. 71 to 116, preferably SEQ ID NOs. 79 and / or SEQ ID NOs. 102, An ex vivo method for modulating gamma-variable 4(Vγ4)T cells, comprising administering them to a cell population containing Vγ4T cells.

[0305] 13. The method as defined in Clause 12, wherein the antibody or fragment thereof includes a VH region containing a CDR3 having at least 80% sequence identity with any one of sequence numbers 2 to 24, such as sequence numbers 10, 4, 14, 15, 17, 19, or 23.

[0306] 14. The method as defined in Clause 12 or Clause 13, wherein the antibody or fragment thereof comprises a VH region including a CDR2 having at least 80% sequence identity with any one of Sequence IDs 48-70, such as Sequence ID 56, 50, 60, 61, 63, 65, or 69.

[0307] 15. The method as defined in any one of the clauses 12 to 14, wherein the antibody or fragment thereof includes a VH region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 71 to 93, such as sequence number 79, 73, 83, 84, 86, 88, or 92.

[0308] 16. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 10, a CDR2 containing the sequence of SEQ ID NO: 56, and a CDR1 containing the sequence of SEQ ID NO: 79, as defined in any one of the provisions of 12 to 15.

[0309] 17. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 4, a CDR2 containing the sequence of SEQ ID NO: 50, and a CDR1 containing the sequence of SEQ ID NO: 73, as defined in any one of clauses 12 to 15.

[0310] 18. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 14, a CDR2 containing the sequence of SEQ ID NO: 60, and a CDR1 containing the sequence of SEQ ID NO: 83, as defined in any one of the clauses 12 to 15.

[0311] 19. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 15, a CDR2 containing the sequence of SEQ ID NO: 61, and a CDR1 containing the sequence of SEQ ID NO: 84, as defined in any one of clauses 12 to 15.

[0312] 20. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 17, a CDR2 containing the sequence of SEQ ID NO: 63, and a CDR1 containing the sequence of SEQ ID NO: 86, as defined in any one of the clauses 12 to 15.

[0313] 21. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 19, a CDR2 containing the sequence of SEQ ID NO: 65, and a CDR1 containing the sequence of SEQ ID NO: 88, as defined in any one of the clauses 12 to 15.

[0314] 22. The antibody or fragment thereof comprises a VH region including a CDR3 containing the sequence of SEQ ID NO: 23, a CDR2 containing the sequence of SEQ ID NO: 69, and a CDR1 containing the sequence of SEQ ID NO: 92, as defined in any one of the clauses 12 to 15.

[0315] 23. The method as defined in any one of the clauses 12 to 22, wherein the antibody or fragment thereof includes a VL region containing a CDR3 having at least 80% sequence identity with any one of sequence numbers 25 to 47, such as sequence number 33, 27, 37, 38, 40, 42, or 46.

[0316] 24. The method as defined in any one of the clauses 12 to 23, wherein the antibody or fragment thereof includes a VL region containing a CDR2 having at least 80% sequence identity with any one of sequences A1 to A23 (in Figure 1), such as sequence A9, A3, A13, A14, A16, A18, or A22.

[0317] 25. The method as defined in any one of the clauses 12 to 26, wherein the antibody or fragment thereof includes a VL region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 94 to 116, such as sequence numbers 102, 96, 106, 107, 109, 111, or 115.

[0318] 26. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 33, a CDR2 containing the sequence of sequence A9, and a CDR1 containing the sequence of sequence number 102, as defined in any one of clauses 12 to 25.

[0319] 27. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 27, a CDR2 containing the sequence of sequence A3, and a CDR1 containing the sequence of sequence number 96, as defined in any one of clauses 12 to 25.

[0320] 28. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 37, a CDR2 containing the sequence of sequence A13, and a CDR1 containing the sequence of sequence number 106, as defined in any one of clauses 12 to 25.

[0321] 29. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 38, a CDR2 containing the sequence of sequence A14, and a CDR1 containing the sequence of sequence number 107, as defined in any one of clauses 12 to 25.

[0322] 30. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 40, a CDR2 containing the sequence of sequence A16, and a CDR1 containing the sequence of sequence number 109, as defined in any one of clauses 12 to 25.

[0323] 31. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 42, a CDR2 containing the sequence of sequence A18, and a CDR1 containing the sequence of sequence number 111, as defined in any one of clauses 12 to 25.

[0324] 32. The antibody or fragment thereof comprises a VL region comprising a CDR3 containing the sequence of sequence number 46, a CDR2 containing the sequence of sequence A23, and a CDR1 containing the sequence of sequence number 115, as defined in any one of clauses 12 to 25.

[0325] 33. Ex. Modulation of gamma variable 4(Vγ4) T cells, comprising administering an anti-Vγ4 antibody or a fragment thereof, which contains an amino acid sequence having at least 80% sequence identity with one of SEQ ID NOs. 117-162 or 261-283, to a cell population containing Vγ4 T cells. vivo method.

[0326] 34. The method as defined in Clause 33, wherein the antibody or fragment thereof includes a VH region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 117 to 139.

[0327] 35. The method as defined in Clause 34, wherein the VH region comprises an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 125, 119, 129, 130, 132, 134, or 138.

[0328] 36. The method as defined in any one of the clauses 33 to 35, wherein the antibody or fragment thereof includes a VL region having an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 140 to 162 or 261 to 283.

[0329] 37. The VL region is, (a) Sequence numbers 148, 142, 152, 153, 155, 157, or 161, or (b) A method as defined in Clause 36, comprising an amino acid sequence having at least 80% sequence identity with any one of Sequence IDs 269, 263, 273, 274, 276, 278, or 282.

[0330] 38. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 125 and a VL region containing the amino acid sequence of SEQ ID NO: 148 or 269, as defined in any one of the provisions of 33 to 37.

[0331] 39. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 119 and a VL region containing the amino acid sequence of SEQ ID NO: 142 or 263, as defined in any one of clauses 33 to 37.

[0332] 40. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 129 and a VL region containing the amino acid sequence of SEQ ID NO: 152 or 273, as defined in any one of clauses 33 to 37.

[0333] 41. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 130, and A method defined in any one of clauses 33 to 37, comprising a VL region containing the amino acid sequence of sequence number 153 or 274.

[0334] 42. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 132 and a VL region containing the amino acid sequence of SEQ ID NO: 155 or 276, as defined in any one of clauses 33 to 37.

[0335] 43. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 134 and a VL region containing the amino acid sequence of SEQ ID NO: 157 or 278, as defined in any one of clauses 33 to 37.

[0336] 44. The antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 138 and a VL region containing the amino acid sequence of SEQ ID NO: 161 or 282, as defined in any one of clauses 33 to 37.

[0337] 45.(a) A VH comprising HCDR1 having sequence number 79, HCDR2 having sequence number 56, and HCDR3 having sequence number 10, wherein the VH may optionally include the VH having sequence number 125. A VL comprising LCDR1 having sequence number 102, LCDR2 having sequence number A9 (in Figure 1), and LCDR3 having sequence number 33, wherein the VL optionally includes sequence number 148 or 269. (b) A VH comprising HCDR1 having sequence number 86, HCDR2 having sequence number 63, and HCDR3 having sequence number 17, wherein the VH may optionally include the VH having sequence number 132. A VL comprising LCDR1 having sequence number 109, LCDR2 having sequence number A16 (Figure 1), and LCDR3 having sequence number 40, wherein the VL optionally includes sequence number 155 or 276. (c) A VH comprising HCDR1 having sequence number 73, HCDR2 having sequence number 50, and HCDR3 having sequence number 4, wherein the VH may optionally be the same as the VH comprising sequence number 119, A VL comprising LCDR1 having sequence number 96, LCDR2 having sequence number A3 (in Figure 1), and LCDR3 having sequence number 27, wherein the VL optionally includes sequence number 142 or 263. (d) A VH comprising HCDR1 having sequence number 83, HCDR2 having sequence number 60, and HCDR3 having sequence number 14, wherein the VH may optionally include the VH having sequence number 129, A VL comprising LCDR1 having sequence number 106, LCDR2 having sequence number A13 (in Figure 1), and LCDR3 having sequence number 37, wherein the VL optionally includes sequence number 152 or 273. (e) A VH comprising HCDR1 having sequence number 84, HCDR2 having sequence number 61, and HCDR3 having sequence number 15, wherein the VH may optionally be the same as the VH comprising sequence number 130, A VL comprising LCDR1 having sequence number 107, LCDR2 having sequence number A14 (Figure 1), and LCDR3 having sequence number 38, wherein the VL optionally includes sequence number 153 or 274. (f) A VH comprising HCDR1 having sequence number 88, HCDR2 having sequence number 65, and HCDR3 having sequence number 19, wherein the VH may optionally be the same as the VH comprising sequence number 134, A VL comprising LCDR1 having sequence number 111, LCDR2 having sequence number A18 (Figure 1), and LCDR3 having sequence number 42, wherein the VL optionally has sequence number 15 The VL including 7 or 278, (g) A VH comprising HCDR1 having sequence number 92, HCDR2 having sequence number 69, and HCDR3 having sequence number 23, wherein the VH may optionally be the same as the VH comprising sequence number 138, A VL comprising LCDR1 having sequence number 115, LCDR2 having sequence number A22 (in Figure 1), and LCDR3 having sequence number 46, wherein the VL optionally includes sequence number 161 or 282. (h) A VH comprising HCDR1 having sequence number 71, HCDR2 having sequence number 48, and HCDR3 having sequence number 2, wherein the VH may optionally be the same as the VH comprising sequence number 117, A VL comprising LCDR1 having sequence number 94, LCDR2 having sequence number A1 (Figure 1), and LCDR3 having sequence number 25, wherein the VL optionally includes sequence number 140 or 261. (i) A VH comprising HCDR1 having sequence number 72, HCDR2 having sequence number 49, and HCDR3 having sequence number 3, wherein the VH may optionally be the same as the VH comprising sequence number 118, A VL comprising LCDR1 having sequence number 95, LCDR2 having sequence number A2 (Figure 1), and LCDR3 having sequence number 26, wherein the VL optionally includes sequence number 141 or 262. (j) A VH comprising HCDR1 having sequence number 74, HCDR2 having sequence number 51, and HCDR3 having sequence number 5, wherein the VH may optionally be the same as the VH comprising sequence number 120. A VL comprising LCDR1 having sequence number 97, LCDR2 having sequence number A4 (Figure 1), and LCDR3 having sequence number 28, wherein the VL optionally includes sequence number 143 or 264. (k) A VH comprising HCDR1 having sequence number 75, HCDR2 having sequence number 52, and HCDR3 having sequence number 6, wherein the VH may optionally be the same as the VH comprising sequence number 121. A VL comprising LCDR1 having sequence number 98, LCDR2 having sequence number A5 (Figure 1), and LCDR3 having sequence number 29, wherein the VL optionally includes sequence number 144 or 265. (l) A VH comprising HCDR1 having sequence number 76, HCDR2 having sequence number 53, and HCDR3 having sequence number 7, wherein the VH may optionally include the VH having sequence number 122. A VL comprising LCDR1 having sequence number 99, LCDR2 having sequence number A6 (Figure 1), and LCDR3 having sequence number 30, wherein the VL optionally includes sequence number 145 or 266. (m) A VH comprising HCDR1 having sequence number 77, HCDR2 having sequence number 54, and HCDR3 having sequence number 8, wherein the VH may optionally be the same as the VH comprising sequence number 123. A VL comprising LCDR1 having sequence number 100, LCDR2 having sequence number A7 (in Figure 1), and LCDR3 having sequence number 31, wherein the VL optionally includes sequence number 146 or 267. (n) A VH comprising HCDR1 having sequence number 78, HCDR2 having sequence number 55, and HCDR3 having sequence number 9, wherein the VH may optionally be the same as the VH comprising sequence number 124. A VL comprising LCDR1 having sequence number 101, LCDR2 having sequence number A8 (in Figure 1), and LCDR3 having sequence number 32, wherein the VL optionally includes sequence number 147 or 268. (o) HCDR1 having sequence number 80, HCDR2 having sequence number 57 and sequence number A VH comprising HCDR3 having number 11, wherein the VH optionally comprises the VH comprising sequence number 126, A VL comprising LCDR1 having sequence number 103, LCDR2 having sequence number A10 (Figure 1), and LCDR3 having sequence number 34, wherein the VL optionally includes sequence number 149 or 270. (p) A VH comprising HCDR1 having sequence number 81, HCDR2 having sequence number 58, and HCDR3 having sequence number 12, wherein the VH may optionally be the same as the VH comprising sequence number 127. A VL comprising LCDR1 having sequence number 104, LCDR2 having sequence number A11 (Figure 1), and LCDR3 having sequence number 35, wherein the VL optionally includes sequence number 150 or 271. (q) A VH comprising HCDR1 having sequence number 82, HCDR2 having sequence number 59, and HCDR3 having sequence number 13, wherein the VH may optionally include the VH having sequence number 128. A VL comprising LCDR1 having sequence number 105, LCDR2 having sequence number A12 (in Figure 1), and LCDR3 having sequence number 36, wherein the VL optionally includes sequence number 151 or 272. (r) A VH comprising HCDR1 having sequence number 85, HCDR2 having sequence number 62, and HCDR3 having sequence number 16, wherein the VH may optionally be the same as the VH comprising sequence number 131, A VL comprising LCDR1 having sequence number 108, LCDR2 having sequence number A15 (Figure 1), and LCDR3 having sequence number 39, wherein the VL optionally includes sequence number 154 or 275. (s) A VH comprising HCDR1 having sequence number 87, HCDR2 having sequence number 64, and HCDR3 having sequence number 18, wherein the VH may optionally be the same as the VH comprising sequence number 133, A VL comprising LCDR1 having sequence number 110, LCDR2 having sequence number A17 (in Figure 1), and LCDR3 having sequence number 41, wherein the VL optionally includes sequence number 156 or 277. (t) A VH comprising HCDR1 having sequence number 89, HCDR2 having sequence number 66, and HCDR3 having sequence number 20, wherein the VH may optionally include the VH having sequence number 135. A VL comprising LCDR1 having sequence number 112, LCDR2 having sequence number A19 (in Figure 1), and LCDR3 having sequence number 43, wherein the VL optionally includes sequence number 158 or 279. (u) A VH comprising HCDR1 having sequence number 90, HCDR2 having sequence number 67, and HCDR3 having sequence number 21, wherein the VH may optionally be the same as the VH comprising sequence number 136, A VL comprising LCDR1 having sequence number 113, LCDR2 having sequence number A20 (Figure 1), and LCDR3 having sequence number 44, wherein the VL optionally includes sequence number 159 or 280. (v) A VH comprising HCDR1 having sequence number 91, HCDR2 having sequence number 68, and HCDR3 having sequence number 22, wherein the VH may optionally be the same as the VH comprising sequence number 137, A VL comprising LCDR1 having sequence number 114, LCDR2 having sequence number A21 (Figure 1), and LCDR3 having sequence number 45, wherein the VL optionally includes sequence number 160 or 281. and / or, (w) A VH comprising HCDR1 having sequence number 93, HCDR2 having sequence number 70, and HCDR3 having sequence number 24, wherein the VH optionally has sequence number 139 Including the aforementioned VH, A VL containing LCDR1 having sequence number 116, LCDR2 having sequence number A23 (Figure 1), and LCDR3 having sequence number 47, wherein optionally, the VL contains one or more of the VLs, including sequence number 162 or 283, an anti-Vγ4 antibody or fragment thereof, An ex vivo method for modulating gamma-variable 4(Vγ4)T cells, comprising administering them to a cell population containing Vγ4T cells.

[0338] 46. ​​The VH region and the VL region are linked by a linker such as a polypeptide linker, as defined in any one of the provisions 33 to 45.

[0339] 47. The linker includes a (Gly4Ser)n type, where n = 1 to 8, as defined in Clause 46.

[0340] 48. The linker is provided in the manner defined in clause 46 or 47, including sequence number 186.

[0341] 49. The linker is provided by the method defined in clause 48, consisting of sequence number 186.

[0342] 50. An ex vivo method for modulating gamma-variable 4(Vγ4)T cells, comprising administering an anti-Vγ4 antibody or a fragment thereof, which contains an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 163-185, to a cell population containing Vγ4T cells.

[0343] 51. The antibody or fragment thereof comprises one amino acid sequence from SEQ ID NOs. 163 to 185, as defined in Clause 50.

[0344] 52. The antibody or fragment thereof is provided by the method defined in Clause 50 or Clause 51, including Sequence ID No. 171, 165, 175, 176, 178, 180, or 184.

[0345] 53. An ex vivo method for modulating gamma-variable 4(Vγ4)T cells, comprising administering an anti-Vγ4 antibody or a fragment thereof, which contains an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 233-255, to a cell population containing Vγ4T cells.

[0346] 54. A method defined in clause 53, comprising any one amino acid sequence from sequence numbers 233 to 255.

[0347] 55. The method defined in clause 53 or clause 54, including sequence numbers 235, 241, 245, 246, or 254.

[0348] 56. An ex vivo method for modulating gamma-variable 4(Vγ4)T cells, comprising administering an anti-Vγ4 antibody or a fragment thereof, comprising administering to a cell population including Vγ4T cells, an anti-Vγ4 antibody or fragment thereof, comprising a heavy chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 284-306 and / or a light chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 307-329.

[0349] 57. A method as defined in Clause 56, comprising a heavy chain amino acid sequence containing any one of SEQ ID NOs. 284-306 and / or a light chain amino acid sequence containing any one of SEQ ID NOs. 307-329.

[0350] 58. The anti-Vγ4 antibody or fragment thereof binds to the same, or essentially the same, epitope as the antibody or fragment thereof as defined in any one of clauses 12 to 57, or A method that conflicts with this, preferably as defined in one of the clauses 1 to 11.

[0351] 59. The antibody or fragment thereof is an scFv, Fab, Fab', F(ab')2, Fv, variable domain (e.g., VH or VL), diabody, minibody, or full-length antibody, as defined in any one of clauses 1 to 58.

[0352] 60. The method as defined in Clause 59, wherein the antibody or a fragment thereof is an scFv or a full-length antibody.

[0353] 61. The method as defined in Clause 60, wherein the antibody or fragment thereof is a full-length antibody such as an IgG1 antibody.

[0354] 62. The method defined in any one of clauses 1 to 61, wherein the antibody or fragment thereof is human.

[0355] 63. The regulation is a method defined in any one of clauses 1 to 62, including the proliferation of Vγ4 T cells.

[0356] 64. The method as defined in Clause 63, which provides a proliferated Vγ4T cell population containing more than 60% Vγ4T cells, such as more than 70% Vγ4T cells.

[0357] 65. The method as defined in any one of clauses 1 to 64, comprising culturing the cell population for at least 5 days.

[0358] 66. The method as defined in any one of clauses 1 to 65, comprising culturing the cell population in the presence of at least one cytokine.

[0359] 67. The cytokine is selected from interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), or a mixture thereof, as defined in Clause 66.

[0360] 68. The method as defined in any one of clauses 1 to 67, comprising culturing the cell population in the presence of IL-2 and / or IL-15.

[0361] 69. The method defined in any one of clauses 65 to 68, wherein the cell population does not come into direct contact with stromal cells and / or epithelial cells during culture.

[0362] 70. The method as defined in Clause 69, wherein the cell population does not come into direct contact with fibroblasts during culture.

[0363] 71. The method defined in any one of clauses 65 to 70, wherein the cell population does not come into direct contact with tumor cells and / or supporting cells during culture.

[0364] 72. The method as defined in any one of clauses 1 to 71, comprising culturing the cell population in serum-free medium.

[0365] 73. The cell population is enriched with T cells prior to administration of the antibody or fragment thereof, by a method defined in any one of clauses 1 to 72.

[0366] 74. The cell population is enriched with γδT cells prior to administration of the antibody or fragment thereof, by a method defined in any one of clauses 1 to 73.

[0367] 75. The method defined in any one of clauses 1 to 74, wherein the cell population is depleted of αβT cells or NK cells prior to administration of the antibody or fragment thereof.

[0368] 76. The cell population is obtained from a hematopoietic sample or a fraction thereof by any one of the methods defined in any one of clauses 1 to 75.

[0369] 77. The hematopoietic sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, lymph node tissue, or fractions thereof, by the method defined in Clause 76.

[0370] 78. The hematopoietic sample comprises peripheral blood mononuclear cells (PBMCs) or low-density mononuclear cells (LDMCs) as defined in Clause 76 or Clause 77.

[0371] 79. The cell population is obtained from a non-hematopoietic tissue sample such as skin, colon, intestine, mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina or other skin, mucous membrane or serosal membrane sample, by any one of the methods defined in any one of Clauses 1 to 75.

[0372] 80. The cell population is obtained from a cancer tissue sample by any one of the methods defined in clauses 1 to 75.

[0373] 81. The cell population is obtained from human or non-human animal tissue by any one of the methods defined in any one of clauses 1 to 80.

[0374] 82. The cell population is isolated from the sample before administration of an anti-Vγ4 antibody or a fragment thereof, by a method defined in any one of clauses 1 to 81.

[0375] 83. A population of Vγ4 T cells obtained by an ex vivo method as defined in any one of clauses 1 to 82.

[0376] 84. A composition comprising the Vγ4T cell population as defined in Article 83.

[0377] 85. A pharmaceutical composition comprising the Vγ4 T cell population as defined in Clause 83, together with a pharmaceutically acceptable diluent or carrier.

[0378] 86. A pharmaceutical composition as defined in Clause 85, for use as a drug.

[0379] 87. A pharmaceutical composition as defined in Clause 86, for use in the treatment of cancer, an infectious disease or an inflammatory disease.

[0380] 88. A method for treating a cancer, infection or inflammatory disease of a subject requiring such treatment, comprising administering a therapeutically effective amount of the Vγ4 T cell population as defined in Clause 83 or the pharmaceutical composition as defined in Clause 85.

[0381] Other features and advantages of the present invention will become apparent from the detailed description provided herein. However, it will be understood that this description and specific examples, while illustrating preferred embodiments of the present invention, are given for illustrative purposes only, as various changes and modifications will become apparent to those skilled in the art. The present invention will now be described using the following non-limiting embodiments. [Examples]

[0382] Example 1. Materials and Method Antigen preparation The design of the soluble γδTCR heterodimers containing the TCRα and TCRβ constant regions used in the following examples was prepared according to Xu et al. (2011) PNAS 108:2414-2419. The Vγ domain or Vδ domain was fused in-frame to the TCRα or TCRβ constant region lacking a transmembrane domain, followed by a leucine zipper sequence or Fc sequence and a histidine tag / linker.

[0383] Expression constructs were transiently transduced into mammalian EXPI HEK293 suspension cells (as single or simultaneous heterodimer transduction). The secreted recombinant proteins were recovered and purified from the culture supernatant by affinity chromatography. To ensure good recovery of monomer antigens, samples were further purified using preparative size exclusion chromatography (SEC). Purified antigens were analyzed for purity by SDS-PAGE and for aggregation by analytical SEC.

[0384] The selected scFv was subcloned into an IgG1 framework using a commercially available plasmid. expi293F suspension cells were transduced with the plasmid for antibody expression. For convenience, unless otherwise specified, the antibodies characterized in these examples refer to IgG1 type antibodies selected as scFv from phage display. However, the antibodies can be any antibody type as described above.

[0385] Antibody purification IgG antibodies were batch purified from the supernatant using protein A chromatography. The quality of the purified IgG was analyzed using ELISA, SDS-PAGE, and SEC-HPLC.

[0386] antigen binding The phage display selectivity output was subcloned into the scFv expression vector pSANG10 (Martin et al. (2006) BMC Biotechnol. 6:46). Soluble scFv was expressed and screened for binding to directly immobilized targets using dissociation-enhanced lantanide fluorescence immunoassay (DELFIA). A hit was defined as a DELFIA signal exceeding 3000 fluorescence units.

[0387] DELFIA ELISA conjugation was also employed to evaluate binding to antibody supernatant or further to purified protein A antibody. Briefly, MaxiSorp plates were coated with 3 μg / ml of antigen BSA or L1 (DV1-GV4), L2 (DV1-GV2), L3 (DV1-GV8), or L4 (DV2-GV4) recombinant TCR antigen. The plates were then washed with PBS, blocked with PBS / skim milk, the test material was added, and incubated at room temperature for 1 hour. Subsequently, the plates were washed with PBS-Tween, DELFIA Eu-N1-anti-human IgG (Perkin Elmer #1244-330) was added at room temperature for 1 hour, followed by further washing, addition of DELFIA enhancement solution (Perkin Elmer #4001-0010), and read using a Pherastar microplate reader.

[0388] D1.3hIgG1 (described in England et al. (1999) J.Immunol. 162:2129-2136) was used as a negative control, and REA173 (Miltenyi) and TS8.2 (ThermoFisher, No. TCR1730) were used as comparator antibodies.

[0389] Antibody experiments using recombinant JRT3-TCR cells Recombinant JRT3-TCR cells used in antibody binding, TCR downregulation, and CD69 upregulation experiments have been previously described (Melandri et al. (2018) Nature Immunology 19(12):1352-1365 and Willcox See et al. (2019)Immunity51(5):813-825.e4).

[0390] For antibody binding experiments, primary staining was performed on either 100,000 untransduced JRT3 control or JRT3-TCR cells in PBS 5% FACS for 30 minutes at 4°C, using the standard 1.0 μg / ml if not specified, or the amount indicated in Figure 6 for each lead antibody, such as 0.08, 0.4, 2, or 10 μg / ml. Secondary staining was then performed using A647 anti-human IgG (Biolegend). Furthermore, BV421 anti-CD3ε (Biolegend) or PE-Cy7IMMU510 anti-γδTCR (Beckman-Coulter) staining was performed as instructed. The cells were then washed twice with PBS 5% FACS, and flow analysis was performed in FACS Canto II 3L.

[0391] In experiments for TCR downregulation / CD69 upregulation, 96 flat-well plates were first pre-coated with 20 μg / ml of secondary antibody, specifically anti-human IgG-Fc (for human D1.3 and Vγ4 antibodies) or anti-mouse IgG (for mouse anti-CD3e or anti-PanTCRgd), added to each well, and then incubated at 37°C for 2 hours. The indicated test antibodies were first diluted to final concentrations of 0.01, 0.1, 1, and 10 μg / ml. Next, 50 μl of each concentration was added to the wells of the pre-coated plates and incubated overnight at 4°C. Then, unbound antibodies were washed twice with PBS, and saturated PBS 5% FCS was added over 1 hour at 37°C. Next, 100,000 cells per well were seeded by rotation at 400 g. Next, the cells were incubated at 37°C for 5 hours, 5% CO2 was added, and the cells were transferred to 96 round-bottom plates for staining. The staining antibodies used included BV421 anti-CD3ε (clone OKT-3Biolegend) diluted 1:400, PE-Cy7 anti-γδTCR (clone IMMU510Beckman Coulter) diluted 1:200, and A647 anti-CD69 (clone FN50Biolegend) diluted 1:200. All staining was performed in 5% FCS in PBS at 4°C for 30 minutes.

[0392] Antibody experiments using primary cell culture cells (PBMCs) 24-well plates were pre-coated by first adding 20 μg / ml (250 μl per well) of anti-human IgG-Fc (Biolegend) to each well, and then incubated at 37°C for 2 hours. Unbound secondary antibodies were washed twice with PBS, and isotype controls (human IgG1, Biolegend) or anti-Vγ4 (clone G4_12) were first diluted to final concentrations of 0.1, 1, and 10 μg / ml. Then, 250 μl of each concentration was added to the wells of the pre-coated plates and incubated overnight at 4°C. Then, unbound antibodies were washed twice with PBS, and saturated PBS 5% FCS was added over 1 hour at 37°C. 100% of the antibodies were added to complete medium (RPMI supplemented with 5% heat-inactivated human AB serum [PAA laboratories], sodium pyruvate [1 mM], and penicillin / streptomycin [ThermoFisher]). 6 500,000 PBMCs, resuspended at a cell / ml ratio, were then added to each well. The cells were then incubated at 37°C in 5% CO2. IL-2 or IL-2+IL-15 (final concentrations of 100 U / ml and 10 ng / ml, respectively) were added after 24 hours, and fresh complete medium supplemented with IL-2 or IL-2+IL-15 was added every 2-3 days. On days 7 and 14 of culture, the cells were transferred to a 96-well round-bottom plate for staining. The staining antibody used was biotin anti-TCRVγ2 / 3 / 4 (diluted to 1 μg / ml). The stains included PE streptavidin (Biolegend) diluted 1:100 (Lone 23D12), BV421 anti-CD3ε (Clone OKT-3 Biolegend) diluted 1:400, PE-Cy7 anti-TCRγδ (Clone IMMU510 Beckman Coulter) and FITC anti-Vδ2 (Clone B6 Biolegend) diluted 1:200, and A647 anti-Vγ4 (Clone G4_18) diluted 1 μg / ml. All staining was performed in 5% FCS in PBS at 4°C for 30 minutes.

[0393] MS-type epitope mapping The CovalX "ultrafast conformational / linear epitope mapping" methodology was employed. First, both protein antigens L1(DV1-GV4) and antibody G4_3(1139_P01_A04) were analyzed for protein integrity and aggregation levels using high-mass MALDI. To measure the binding epitope of the L1(DV1-GV4) / G4_3 complex at high resolution, the complex was incubated with a deuterated crosslinking agent and subjected to multi-enzyme proteolysis using trypsin, chymotrypsin, Asp-N, elastase, and thermolysin. After enriching the crosslinked peptides, the samples were analyzed using a high-resolution mass spectrometer (nLC-LTQ-Orbitrap MS), and the generated data were analyzed using XQuest and Stavrox software.

[0394] γδT cell binding assay Antibody binding to γδT cells can be tested by incubating a fixed concentration of purified antibody with 250,000 γδT cells. This incubation can be performed under blockage conditions, such as by adding a huFc fragment or Ig, to prevent nonspecific binding of the antibody via the Fc receptor. Detection can be performed by adding a secondary fluorescent dye-conjugated antibody against human IgG1. For negative controls, cells can be prepared with a) isotype antibody only (recombinant human IgG), b) fluorescent dye-conjugated anti-human IgG antibody only, and c) a combination of a) and b). Control wells of completely unstained cells can also be prepared and analyzed. As a positive control, purified mouse monoclonal IgG2 anti-human CD3 antibody can be used at two different concentrations and stained with a fluorescent dye-conjugated goat anti-mouse secondary antibody. The assay is acceptable if the mean fluorescence intensity in the FITC channel of the low-concentration positive control is at least 10 times that of the highest-concentration negative control.

[0395] SPR analysis SPR analysis can be performed using MASS-2 instruments equipped with large amine chips (both manufactured by Sierra Sensors, Germany). 15 nM IgG is captured via protein G to the large amine chip (100 nM at TS8.2). Using the following parameters, 180 seconds of association, 600 seconds of dissociation, a flow rate of 30 μL / min, and electrophoresis buffer PBS + 0.02% Tween20, L1 (DV1-GV4) antigen can flow over cells in a 1:2 dilution series from 2000 nM to 15.625 nM. All experiments were performed at room temperature using MASS-2 instruments. Steady-state fitting can be determined according to Langmuir 1:1 binding using the software SierraAnalyzer3.2.

[0396] γδTCR Downregulation and Degranulation Assay THP-1 (TIB-202™, ATCC) target cells loaded with or unloaded with the test antibody can be labeled with CellTracker™ Orange CMTMR (ThermoFisher, C2927) and incubated with γδT cells in a 2:1 ratio in the presence of CD107a antibody (anti-human CD107a BV421 (clone H4A3) BD Biosciences 562623). After 2 hours of incubation, flow cytometry can be used to evaluate the surface expression of γδTCR in γδT cells (to measure TCR downregulation) and CD107a expression (to measure degranulation).

[0397] Death assay The effects of test antibodies on gamma delta T cell-mediated death activity and γδ T cell death activity can be obtained by flow cytometry. After co-culturing γδ T cells and THP-1 cells labeled with CellTracker® Orange CMTMR (ThermoFisher, C2927) in vitro at a 20:1 ratio for 4 hours (with or without antibody loading), viability of target THP-1 cells can be distinguished by staining with Viability Dye eFluor® 520 (ThermoFisher, 520 65-0867-14). During sample acquisition, target cells can be gated for CellTracker® Orange CMTMR positivity, and cell death can be tested based on Viability Dye uptake. Cells double-positive for both CMTMR and eFluor® 520 can be recognized as dead target cells. γδ T cell death activity is presented as the percentage of dead target cells.

[0398] Example 2. Antigen Design Gamma delta (γδ) T cells are polyclonal, possessing CDR3 polyclonality. To avoid situations where the produced antibody is selected against the CDR3 sequence (since the CDR3 sequence differs from TCR clone to clone), the antigen design involved maintaining a consistent CDR3 in different forms. This design aimed to produce antibodies that recognize sequences within the variable domain, which are encoded in the germline and therefore identical in all clones, thereby providing antibodies that recognize a broader subset of γδ T cells.

[0399] Another important aspect of the antigen preparation process is designing antigens suitable for expression as proteins. γδTCR is a complex protein containing heterodimers with interchain and intrachain disulfide bonds. Soluble TCR antigens for use in phage display selection were prepared using leucine zipper (LZ) and Fc forms. Both LZ and Fc forms were well expressed and successfully displayed TCRs (particularly heterodimeric TCRs, e.g., Vδ1Vγ4).

[0400] The CDR3 sequence from a publicly available database entry for γδTCR was found to be expressed similarly to the protein (RSCB Protein Databank entry: 4MNH). Therefore, this was selected for antigen preparation.

[0401] Antigens containing gamma-variable 4-chains were expressed as LZ heterodimers (i.e., in combination with different delta-variable chains—for example, DV1-GV4, a heterodimer consisting of one delta-variable chain and one gamma-variable 4-chain [referred to as "L1"] and DV2-GV4, a heterodimer consisting of two delta-variable 2-chains and one gamma-variable 4-chain [referred to as "L4"]), as well as as Fc heterodimers or homodimers (i.e., in combination with another gamma-variable 4-chain—GV4-GV4, a homodimer consisting of two gamma-variable 4-chains [referred to as "Fc4 / 4"]). All gamma-variable 4-chains of the antigen contained 4MNH CDR3. Another set of γδTCR antigens using a similar format were designed to contain different gamma variable chains (such as gamma variable 2 and gamma variable 8) and were used to deselect antibodies with nonspecific or off-target binding (e.g., a heterodimer consisting of DV1-GV2, delta variable 1 chain and gamma variable 2 chain [referred to as "L2"] or a heterodimer consisting of DV1-GV8, delta variable 1 chain and gamma variable 8 chain [referred to as "L3"]). These antigens were also designed to contain 4MNH CDR3, ensuring that antibodies binding to the CDR3 region were also reliably deselected.

[0402] Example 3. Phage Display Phage display selection involves the heterodimer LZ TCR type in process 1 and process 2. Either process 1 was performed on a human scFv library, and heterodimer LZ TCRs were deselected in both processes. Alternatively, process 1 was performed using homodimer Fc fusion TCRs with human IgG1Fc deselected, followed by process 2 with heterodimer LZTCRs with heterodimer LZ TCRs deselected (see Table 1).

[0403] [Table 2]

[0404] Selection was performed in the solution phase using 100 nM biotinylated protein. Deselection was performed using 1 μM non-biotinylated protein.

[0405] Example 4. Selection of Antibodies The hits obtained in Example 3 were sequenced (using standard methods known in the art). 130 unique clones were identified, showing unique combinations of VH and VL CDR3. Of these 130 unique clones, 129 showed unique VHCDR3 and 116 showed unique VLCDR3.

[0406] Unique clones were rearranged, and their specificity was analyzed by ELISA (DELFIA). A panel of 42 unique human scFv binders that bind to TRGV4 but not to TRGV2 or TRGV8 was identified from the selected samples.

[0407] To assist in future clone selection, affinity rankings of selected binders were included. Many binders exhibited nanomolar affinities and reacted with 25–100 nM biotinylated antigens (L1). A few binders showed strong reactions with 5 nM antigens, suggesting potential single-digit nanomolar affinities. Some binders did not react with 100 nM antigens, exhibiting micromolar affinities.

[0408] The goal was to include as many germline cells and as many different CDR3s as possible in order to select clones that would proceed to IgG conversion. Furthermore, sequence tendencies such as glycosylation, integrin binding sites, CD11c / CD18 binding sites, and unpaired cysteine ​​were avoided. In addition, a variety of affinities were included. The clones selected for IgG conversion are shown in Figure 1. The ELISA binding results (fluorescence units (FU) values) are shown in Figure 2A. The results show that all 23 antibodies exhibit the desired gamma 4 chain specific profile regardless of the partner delta chain. The same data is also shown in Figure 2B, which further shows the increasing multiplicity of binding of each clone to human Vγ4 chain versus human Vγ2 chain. The increasing multiplicity of binding of human Vγ4 chain versus human Vγ2 chain ranged from 80-fold (clone G4_26) to 98387-fold (clone G4_18).

[0409] Antibody binding experiments were performed using recombinant Jurkat (JRT3-hu17) cells. A comparison of ELISA and flow cytometry data is shown in Figure 3A. Antibody clones that were confirmed to bind to both the DV1-GV4 antigen (Y axis) and JRT3-hu17 cells (X axis) via lfia ELISA were selected for further study.

[0410] Example 5. Study on Vγ4 antibody binding The ability of the antibodies selected in Example 4 to stain Vγ4TCRs with different CDR3 sequences (hu17 vs. hu20, both Vγ4Vδ1) or delta chains (hu20γ / huPBδ, Vγ4Vδ2;LES, Vγ4Vδ5) was studied. The results are shown in Figure 4A, showing that all antibodies tested showed significantly increased binding to one or more Vγ4TCRs used in the experiment compared to the D1.3 isotype control. In particular, five exemplary antibodies (G4_3, G4_12, G4_16, G4_18, and G4_27) bound to all Vγ4TCRs expressed in this experiment and showed significantly enhanced binding signals compared to the D1.3 isotype control, regardless of the CDR3 sequence or the partner's delta chain. As an example, Figure 4B shows examples of flow data for the two antibodies in this experiment to illustrate the difference between G4_3 binding (positive staining for all Vγ4TCRs) and G4_4 binding (positive staining for both hu17 and LES, but reduced staining for both hu20 [a different CDR3 sequence compared to hu17] and hu20g / huPBd [Vγ4Vδ2]).

[0411] Example 6. Epitope mapping using chimeric hu17TCR hu17 is a Vγ4 / Vδ1 TCR cloned from BTNL3+8-reactive human colorectal intraepithelial lymphocytes by single-cell PCR (as described in Melandri et al. (2018) Nat.Immunol. 19:1352-1365). Different chimeric hu17 TCR constructs were prepared, as summarized in Figure 5A. These constructs are derived from hu17 and are all from Melandri et al. (2018) Nat.Immunol. 19:1352-1365 and Willcox et al. This is described in al. (2019) Immunity 51:813-825 (both incorporated herein by reference).

[0412] Next, antibody binding was studied by flow cytometry against chimeric hu17 TCRs expressed in JRT3 cells. Figure 5B shows a table summarizing the reactivity of each antibody to the shown chimeric TCR constructs. The results highlight the relative binding specificity of the individual antibodies shown to individual TCRs expressed in JRT3 cells. When the hu17 TCR construct containing the Vγ2 sequence was used on the HV4 region, no staining or reduction in staining was observed, indicating that antibodies G4_3, G4_12, G4_16, G4_18, and G4_27 all specifically bind to or around the HV4 region.

[0413] Figure 5C shows an example of epitope mapping flow data to explain the different binding signals observed in this experiment. This example and this epitope mapping approach demonstrate G4_12 binding to various recombinant chimeric TCRs. Initially, G4_12 shows strong binding to the initiating hu17 TCR (leftmost panel). Strong binding is also observed to hu17 when the CDR1+2 sequence is replaced in-frame with a CDR equivalent to Vγ2 (center left panel) or when hu17 is HV4 modified to the Vγ2 sequence DG>YA (center right panel). However, a significant decrease in binding by G4_12 was observed with alternative amino acid substitutions of Vγ4 to Vγ2 (DGKM>YANL, center panel) or KM>NL (rightmost panel), respectively. Therefore, in this case, the epitope recognized by G4_12 is the HV4 region (amino acids 67-82 of SEQ ID NO: 1).

[0414] [ka]

[0415] It is located at ) and is greatly affected by changing the underlined K and M residues found at this position in the Vγ2 HV4 region to equivalent residues.

[0416] Example 7. Titration of studied antibodies in staining and functional assays. Figure 6A shows the titration results of the studied antibodies for staining and analysis by flow cytometry on JRT3-hu17 cells (concentrations ranging from 0.08 to 10 μg / mL, 5-fold dilution). Untransduced JRT3 cells (without TCR) were used as a negative control. The results indicate that all antibodies were able to bind to JRT3 cells expressing Vγ4TCR.

[0417] Next, functional assays were performed by studying TCR turnover and CD69 upregulation with titration antibodies against turnover induced by anti-CD3ε binding or anti-pan-TCRγδ antibodies. Figures 6B and 6C show the results for five antibodies, and Table 2 summarizes the results of a broad selection of antibodies within the original cohort.

[0418] [Table 3]

[0419] All antibodies listed in Table 2 have been shown to bind to the Vγ4 chain of the γδTCR. However, as shown in the table, some of these antibodies can activate the Vγ4TCR as measured by downregulation of the Vγ4TCR and / or increased CD69 expression (indicated as "+", "++" or "+++", where "+++" signifies the highest relative level of activation), while others do not show any apparent ability to activate the Vγ4TCR (indicated as "-").

[0420] Example 8. Epitope mapping based on MS To determine the epitopes of antigen / antibody complexes with high resolution, protein complexes were incubated with a deuterated crosslinking agent and subjected to multi-enzyme cleavage. After enriching the crosslinked peptides, the samples were analyzed using a high-resolution mass spectrometer (nLC-LTQ-Orbitrap MS), and the generated data were analyzed using XQuest (version 2.0) and Stavrox (version 3.6) software.

[0421] Deuterated protein complex L1(DV1-GV4) / 1139_P01_A04 d0 After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis on day 12, 11 cross-linked peptides were detected between L1 (DV1-GV4) and antibody 1139_P01_A04 (G4_3) by nLC orbitrap-type MS / MS analysis. The results of epitope mapping are shown in Table 3.

[0422] [Table 4]

[0423] This epitope mapping data correlates with the aforementioned experiment and indicates that this antibody binds to the HV4 region of the γ4 chain.

[0424] Example 9. Targeting and regulation of primary Vγ4-positive cells with anti-Vγ4 antibody. Primary Vγ4 derived from skin, blood, and intestines, including cells from samples of healthy and diseased patients. + Further experiments were conducted to demonstrate the cell-targeting anti-Vγ4 antibody.

[0425] Skin-derived primary Vγ4 + Binding to cells First, the original Vγ4 was grown from the skin of two individual donors. + Anti-Vγ4 antibodies were tested for binding to T cells. Skin samples were prepared by removing subcutaneous fat and using 3 mm biopsy punches to create multiple punches. The punches were placed on a carbon matrix grid and placed in wells of G-REX6 (Wilson Wolf). Each well was filled with complete isolation medium containing AIM-V medium (Gibco, Life Technologies), CTS immunoserum replacement (Life Technologies), IL-2, and IL-15. For the first 7 days of culture, amphotericin B (Life Technologies) was used. Complete isolation medium containing ("+AMP") (Technologies) was used. The medium was changed every 7 days by gently aspirating the upper medium and replacing it with 2X complete isolation medium (without AMP) to avoid disturbing the cells at the bottom of the plate or bioreactor. After culturing for more than 3 weeks, before collection, the released cells obtained were then cultured with recombinant IL-2, IL-4, IL-15, and IL-21 in a new tissue culture vessel and fresh medium (e.g., AIM-V medium or TexMAX medium (Miltenyi)). Subsequently, αβT cells present in the culture were also removed with the help of an αβT cell depletion kit and associated protocol, such as that provided by Miltenyi. For further details, see International Patent No. WO2020 / 095059.

[0426] After isolation, γδT cells were first stained with a viability dye in the presence of Fc block at 4°C for 20 minutes. Next, γδT cells were incubated with exemplary anti-Vγ4 antibody (0.046–100 μg / ml) or isotype control (IgG1 anti-respiratory polynuclear virus (RSV) antibody) at fixative concentrations at 4°C for 30 minutes. Detection was performed by adding a secondary fluorescent dye-complex antibody against human IgG1 (IS11-12E4.23.20). The cells were then fixed and acquired using a MACSQuant16 flow cytometer. The cells were then subjected to a single live IgG1(Vγ4) + It was gated as follows. The data shown is Vγ4 + This is the median fluorescence intensity (MFI) of the secondary detection antibody that was bound to cells and detected.

[0427] The results are shown in Figure 7A. These data indicate that all tested anti-Vγ4 antibodies dose-dependently reacted with primary cutaneous Vγ4. + We confirmed that it can bind to T cells. Binding was not observed in isotype controls.

[0428] Primary Vγ4 derived from peripheral blood mononuclear cells (PBMCs) + binding to In short, human PBMCs (Lonza, product code CC-2702) were first stained with viability dye in the presence of Fc blocks at 4°C for 20 minutes. Next, the cells were incubated with 10 μg / ml anti-Vγ4 antibody or isotype control (RSV) at 4°C for 30 minutes, washed, and then extracellularly stained with anti-Vδ1 (REA173), anti-Vδ2 (REA771), anti-γδ (REA591), and anti-human IgG1 (IS11-12E4.23.20) at 4°C for 20 minutes. The cells were then fixed and acquired using a MACSQuant16 flow cytometer. The cells were then stained with a single live γδ + Vδ2 - IgG1(Vγ4) + It was gated as such.

[0429] The results are shown in Figure 7B. The data shown are for γδ detected using individual antibodies conjugated with a combined secondary anti-human IgG1 antibody. + Vδ2 - Cellular Vγ4 + This is the percentage of cells. These data are derived from primary blood-derived anti-Vγ4 + This highlights the ability of virtually all anti-Vγ4 antibodies to bind to T cells. Antibodies G4_23, G4_3, G4_12, G4_18, and G4_20 were used to detect the strongest signals.

[0430] Primary Vγ4 of intestinal-derived intraepithelial lymphocytes (IELs) obtained from colorectal cancer (CRC) patients. + Binding to cells In this experiment, human CRC tumor biopsies were shipped fresh and processed upon receipt. The biopsies were approximately 2 mm in size. 2 Tumor-infiltrating lymphocytes (TILs), cut into fragments of up to 2 mm in size, were obtained using an application of the method first described by Kupper and Clarke (Clarke et al., 2006, J. Invest. Dermatol. 126, 1059-1070). Specifically, up to four 2 mm fragments were obtained. 2Biopsies were placed in 9mm x 9mm x 1.5mm Cellfoam matrix, with one matrix per well in a 24-well plate. Next, biopsies were performed in 2 ml of Iskoff-modified Dulbecco's medium (IMDM) supplemented with 4% human plasma, β-mercaptoethanol (50 μM), penicillin (100 U / ml), streptomycin (100 μg / ml), gentamicin (20 μg / ml), metronidazole (1 μg / ml), amphotericin B (2.5 μg / ml), HEPES (10 mM), sodium pyruvate (1 mM), MEM non-essential amino acid solution (1X), and IL-15 (20 ng / ml, Miltenyi Biotech). 1 ml of medium was aspirated every 3 days and replaced with 1 ml of complete medium containing 2× concentrated IL-15. After 10 days, TILs were collected, filtered through a 70 μM nylon cell filter, centrifuged at 300 x g for 5 minutes, and resuspended in complete medium to determine the phenotype. TILs were first stained with viability dyes at 4°C for 20 minutes in the presence of Fc blocks. Next, cells were incubated with 10 μg / ml anti-Vγ4 antibody or isotype control (RSV) at 4°C for 30 minutes, washed, and extracellularly stained with anti-Vδ1 (REA173), anti-γδ (REA591), and anti-human IgG1 (IS11-12E4.23.20) at 4°C for 20 minutes. Cells were then fixed and acquired using a MACSQuant16 flow cytometer. Cells were then stained with a single live γδ + IgG1 + (Vγ4) + It was gated as such.

[0431] The results are shown in Figure 7C. The data shown are primary intestinal Vγ4 detected via a combined secondary anti-human IgG1 antibody. + This is a FACS plot showing the binding of anti-Vγ4 antibodies G4_3, G4_12, and G4_18 to cells. The data were obtained from CRC tumor tissue. + This demonstrates the ability of the antibody of the present invention to bind to cells.

[0432] Detection of human gut-derived γδT cells conferred by anti-Vγ4 antibody and TCR downregulation. Further experiments were conducted to study the regulation of human gut-derived γδ T cells conferred by anti-Vγ4 antibodies. In these experiments, normal adjacent tissue (NAT) biopsies from the colon of CRC patients were shipped fresh and processed upon receipt to obtain single-cell suspensions. Specifically, the tissue was divided into approximately 2 mm sections. 2 Up to 1 g of tissue was placed in a Miltenyi C tube with 4.7 ml of RPMI containing the manufacturer's recommended concentration of enzymes from the Miltenyi Tumor Dissociation Kit, other than enzyme R, which was finely chopped to the size of the tissue and used at a concentration of 0.2X to prevent cleavage of related cell surface molecules. The C tube was placed in a gentleMACS® Octo Dissociator fitted with a heating block. Program 37C_h_TDK_1 was selected for the dissociation of soft tumors. After 1 hour, the digest was filtered through a 70 μM filter and the enzyme activity was quenched by adding complete IMDM containing 4% human plasma. The cells were then washed twice and resuspended in complete IMDM for counting. At this point, the cells were seeded for stimulation with anti-Vγ4 antibody or used to determine the phenotype.

[0433] In a series of experiments, Vγ4 in the intestinal digested product before stimulation with anti-Vγ4 antibodies + The phenotype of γδT cells was measured. Briefly, cells were stained with viability dyes for 20 minutes at 4°C in the presence of Fc blocks. Next, the cells were incubated with 10 μg / ml G4_18 clones at 4°C for 30 minutes, washed, and extracellularly stained with anti-Vδ1 (REA173), anti-γδ (REA591), anti-CD69 (REA824), anti-CD103 (Ber-Act8), and anti-human IgG1 (IS11-12E4.23.20) at 4°C for 20 minutes. The cells were then fixed and acquired using a MACSQuant16 flow cytometer. As shown in Figure 7D, 1.4% of living single cells were Vδ1 + Of these, 44.2% were paired with Vγ4, and as expected for γδT cells from the gut, all of them displayed markers of intestinal tissue retention (CD69). + CD103 +The antibodies described herein, in this case the exemplary antibody G4_18, are Vγ4 isolated from human intestinal tissue. + These results confirm that it can be used to specifically detect γδT cells.

[0434] The following series of experiments measured the effects of stimulating cells with anti-Vγ4 antibodies. 2x10 6 Single cells were seeded in each well of a 48-well plate and stimulated with G4_12, G4_18, or RSVIgG1 isotype control antibodies in the presence of IL-15 at a concentration of 2 ng / ml. Intraepithelial lymphocytes (IELs) isolated by enzymatic digestion were analyzed by flow cytometry 24 hours after mAb stimulation. After 24 hours of stimulation, cells were stained with viability dye at 4°C for 20 minutes in the presence of Fc block. Next, cells were stained extracellularly for γδTCR (REA591), fixed, and acquired by MACSQuant16 flow cytometry. Living single cells were stained for γδTCR + Gated as such. Figure 7E shows the γδTCR downregulation induced after 24-hour stimulation in G4_12 and G4_18 clones compared to RSV isotype controls, along with representative FACS plots. Both anti-Vγ4 antibodies, G4_12 and G4_18, induced γδTCR downregulation compared to RSV isotype controls, with the greatest downregulation observed in G4_12.

[0435] Example 10. Further experiments to measure the binding affinity (KD) to human Vγ4 as measured by surface plasmon resonance (SPR) of an exemplary anti-Vγ4 antibody of the present invention. Furthermore, in addition to the SPR binding experiment described in Example 4 (method described in Example 1) regarding the scFv binder, additional experiments were performed to measure the binding affinity of the selected example clone to the human Vγ4 chain when the clone was expressed as a complete IgG1 monoclonal antibody.

[0436] In short, the binding affinity of the antibody to the target (i.e., the human Vγ4 chain of the γδTCR) was established by SPR analysis using a Reichert 4SPR instrument (Reichert Technologies). The antigen (L1(DV1-GV4)) was placed on a carboxymethyl dextran tip (Reichert Technologies). Antibodies were bound at 10 ug / ml, resulting in an increase of approximately 750 uRIU from baseline. Antibodies were flowed over cells in 1:2 dilution systems ranging from 500 nM to 31.25 nM with the following parameters: association time of 180 seconds, dissociation time of 300 seconds, flow rate of 25 μL / min, and electrophoresis buffer PBS + 0.05% Tween20. All experiments were performed at room temperature, and samples were kept at 4°C before flowing over the chip. Steady-state fitting was determined according to Langmuir 1:1 binding using TraceDrawer software (Reichert Technologies).

[0437] The results are shown in Table 4, representing the average of two experiments per antibody (unless otherwise specified).

[0438] [Table 5]

[0439] As expected, the range of binding affinity was determined, and therefore, it became possible to select a specific antibody for a particular situation depending on the required binding affinity. In particular, as shown, the binding affinity was in the range of approximately 260 nM to 2.8 nM. This was consistent with the scFv experiment described in Example 4.

[0440] Example 11. Use of Vγ4-specific antibodies to increase the number of primary human Vγ4 T cells The antibody exhibiting the highest stimulating activity against JRT3-hu17 cells (clone G4_12, Figure 5B, C) was further tested for its ability to stimulate primary Vγ4+ T cells. Compared to isotype controls, the increase in the percentage of Vγ4 T cells in PBMC medium after plate-bound stimulation with G4_12 was analyzed by flow cytometry using a panel of antibodies including A647-complexed anti-Vγ4 clone G4_18, and is shown in Figure 8. The percentage of Vg4-positive cells in the presence of G4_12 antibody on days 7 and 14 was greater than in the medium with isotype controls.

[0441] [Table 6-1]

[0442] [Table 6-2]

[0443] [Table 6-3]

[0444] [Table 6-4]

[0445] [Table 6-5]

[0446] [Table 6-6]

[0447] [Table 6-7]

[0448] [Table 6-8]

[0449] Table 6-9

[0450] Table 6-10

[0451] Table 6-11

[0452] Table 6-12

[0453] Table 6-13

[0454] Table 6-14

[0455] Table 6-15

[0456] Table 6-16

[0457] Table 6-17

[0458] Table 6-18

[0459] Table 6-19

[0460] Table 6-20

[0461] Table 6-21

[0462] Table 6-22

[0463] Table 6-23

[0464] Table 6-24

[0465] Table 6-25

[0466] Table 6-26

[0467] Table 6-27

[0468] Table 6-28

[0469] Table 6-29

[0470] Table 6-30

[0471] Table 6-31

[0472] Table 6-32

[0473] Table 6-33

[0474] Table 6-34

[0475] Table 6-35

[0476] Table 6-36

[0477] Table 6-37

[0478] Table 6-38

[0479] Table 6-39

[0480] Table 6-40

[0481] Table 6-41

[0482] Table 6-42

[0483] Table 6-43

[0484] Table 6-44

[0485] Table 6-45

[0486] Table 6-46

[0487] Table 6-47

[0488] Table 6-48

[0489] Table 6-49

[0490] Table 6-50

[0491] Table 6-51

[0492] Table 6-52

[0493] Table 6-53

[0494] Table 6-54

[0495] Table 6-55

[0496] Table 6-56

[0497] Table 6-57

[0498] Table 6-58

[0499] Table 6-59

[0500] Table 6-60

[0501] Table 6-61

[0502] Table 6-62

[0503] Table 6-63

[0504] Table 6-64

[0505] Table 6-65

[0506] Table 6-66

[0507] Table 6-67

[0508] Table 6-68

[0509] Table 6-69

[0510] Table 6-70

[0511] Table 6-71

[0512] Table 6-72

[0513] Table 6-73

[0514] Table 6-74

[0515] Table 6-75

[0516] Table 6-76

[0517] Table 6-77

[0518] Table 6-78

[0519] Table 6-79

[0520] Table 6-80

[0521] Table 6-81

[0522] Table 6-82

Claims

1. An ex vivo method for modulating Vγ4 T cells, comprising administering an antibody or fragment thereof that specifically binds to the gamma variable 4 (Vγ4) chain of the γδ T cell receptor (TCR) but does not bind to the gamma variable 2 (Vγ2) chain of the γδ TCR to a cell population containing Vγ4 T cells.

2. The method according to claim 1, wherein the Vγ4 chain of the γδTCR is human Vγ4 and the Vγ2 chain of the γδTCR is human Vγ2.

3. The method according to claim 1 or 2, wherein the antibody or fragment thereof binds to an epitope of the Vγ4 chain of the γδTCR, which includes one or more amino acid residues within the amino acid region 67-82 of SEQ ID NO:

1.

4. The method according to claim 3, wherein the epitope comprises at least one of the amino acid residues 71, 73, 75, 76, and 79 of SEQ ID NO:

1.

5. The method according to any one of claims 1 to 4, wherein the epitope includes or consists of K76 and / or M80 of Sequence ID No.

1.

6. The method according to any one of claims 1 to 5, wherein the epitope is an activating epitope for γδ T cells.

7. The method according to claim 6, wherein the binding of the activated epitope (i) downregulates the γδTCR, (ii) activates degranulation of the γδT cells, (iii) activates death mediated by γδT cells, and / or (iv) activates or increases cell signaling mediated by the Vγ4 chain.

8. CDR3 includes one of sequence numbers 2 to 47, preferably a sequence having at least 80% sequence identity with sequence number 10 and / or sequence number 33. CDR2 includes one of sequence numbers 48-70 and sequences A1-A23 (in Figure 1), preferably sequence number 56 and / or sequence A9, and / or a sequence having at least 80% sequence identity. An anti-gamma variable 4 (Vγ4) antibody or a fragment thereof, comprising one or more CDR1 sequences having at least 80% sequence identity with any one of SEQ ID NOs: 71 to 116, preferably SEQ ID NO: 79 and / or SEQ ID NO: 102, An ex vivo method for modulating Vγ4T cells, comprising administering a cell population containing Vγ4T cells.

9. The method according to claim 8, wherein the antibody or fragment thereof includes a VH region comprising a CDR3 having at least 80% sequence identity with any one of sequence numbers 2 to 24, such as sequence numbers 10, 4, 14, 15, 17, 19, or 23.

10. The method according to claim 8 or 9, wherein the antibody or fragment thereof includes a VL region containing a CDR3 having at least 80% sequence identity with any one of sequence numbers 25 to 47, such as sequence number 33, 27, 37, 38, 40, 42, or 46.

11. An ex vivo method for modifying Vγ4 T cells, comprising administering an anti-gamma variable 4 (Vγ4) antibody or a fragment thereof, which has at least 80% sequence identity with any one of sequence numbers 117-162 or 261-283, to a cell population including Vγ4 T cells.

12. (a) A VH comprising HCDR1 having sequence number 79, HCDR2 having sequence number 56, and HCDR3 having sequence number 10, wherein the VH may optionally be the same as the VH comprising sequence number 125, A VL including LCDR1 having sequence number 102, LCDR2 having sequence number A9 (in Figure 1), and LCDR3 having sequence number 33, wherein the VL optionally includes sequence number 148 or 269. (b) A VH comprising HCDR1 having sequence number 86, HCDR2 having sequence number 63, and HCDR3 having sequence number 17, wherein the VH may optionally be the same as the VH comprising sequence number 132. A VL including LCDR1 having sequence number 109, LCDR2 having sequence number A16 (in Figure 1), and LCDR3 having sequence number 40, wherein the VL optionally includes sequence number 155 or 276. (c) A VH comprising HCDR1 having sequence number 73, HCDR2 having sequence number 50, and HCDR3 having sequence number 4, wherein the VH may optionally be the same as the VH having sequence number 119, A VL including LCDR1 having sequence number 96, LCDR2 having sequence number A3 (in Figure 1), and LCDR3 having sequence number 27, wherein the VL optionally includes sequence number 142 or 263. (d) A VH comprising HCDR1 having sequence number 83, HCDR2 having sequence number 60, and HCDR3 having sequence number 14, wherein the VH may optionally be the same as the VH having sequence number 129, A VL including LCDR1 having sequence number 106, LCDR2 having sequence number A13 (in Figure 1), and LCDR3 having sequence number 37, wherein the VL optionally includes sequence number 152 or 273. (e) A VH comprising HCDR1 having sequence number 84, HCDR2 having sequence number 61, and HCDR3 having sequence number 15, wherein the VH may optionally be the same as the VH having sequence number 130, A VL including LCDR1 having sequence number 107, LCDR2 having sequence number A14 (in Figure 1), and LCDR3 having sequence number 38, wherein the VL optionally includes sequence number 153 or 274. (f) A VH comprising HCDR1 having sequence number 88, HCDR2 having sequence number 65, and HCDR3 having sequence number 19, wherein the VH may optionally be the same as the VH comprising sequence number 134. A VL including LCDR1 having sequence number 111, LCDR2 having sequence number A18 (in Figure 1), and LCDR3 having sequence number 42, wherein the VL optionally includes sequence number 157 or 278. (g) A VH comprising HCDR1 having sequence number 92, HCDR2 having sequence number 69, and HCDR3 having sequence number 23, wherein the VH may optionally be the same as the VH comprising sequence number 138. A VL including LCDR1 having sequence number 115, LCDR2 having sequence number A22 (in Figure 1), and LCDR3 having sequence number 46, wherein the VL optionally includes sequence number 161 or 282. (h) A VH comprising HCDR1 having sequence number 71, HCDR2 having sequence number 48, and HCDR3 having sequence number 2, wherein the VH may optionally be the same as the VH having sequence number 117. A VL including LCDR1 having sequence number 94, LCDR2 having sequence number A1 (in Figure 1), and LCDR3 having sequence number 25, wherein the VL optionally includes sequence number 140 or 261. (i) HCDR1 having sequence number 72, HCDR2 having sequence number 49 and sequence number A VH containing HCDR3 having number 3, wherein the VH optionally contains the VH having sequence number 118, A VL including LCDR1 having sequence number 95, LCDR2 having sequence number A2 (in Figure 1), and LCDR3 having sequence number 26, wherein the VL optionally includes sequence number 141 or 262. (j) A VH comprising HCDR1 having sequence number 74, HCDR2 having sequence number 51, and HCDR3 having sequence number 5, wherein the VH may optionally include the VH having sequence number 120. A VL including LCDR1 having sequence number 97, LCDR2 having sequence number A4 (in Figure 1), and LCDR3 having sequence number 28, wherein the VL optionally includes sequence number 143 or 264. (k) A VH comprising HCDR1 having sequence number 75, HCDR2 having sequence number 52, and HCDR3 having sequence number 6, wherein the VH may optionally be the same as the VH comprising sequence number 121. A VL including LCDR1 having sequence number 98, LCDR2 having sequence number A5 (in Figure 1), and LCDR3 having sequence number 29, wherein the VL optionally includes sequence number 144 or 265. (l) A VH comprising HCDR1 having sequence number 76, HCDR2 having sequence number 53, and HCDR3 having sequence number 7, wherein the VH may optionally be the same as the VH comprising sequence number 122. A VL including LCDR1 having sequence number 99, LCDR2 having sequence number A6 (in Figure 1), and LCDR3 having sequence number 30, wherein the VL optionally includes sequence number 145 or 266. (m) A VH comprising HCDR1 having sequence number 77, HCDR2 having sequence number 54, and HCDR3 having sequence number 8, wherein the VH may optionally be the same as the VH comprising sequence number 123. A VL including LCDR1 having sequence number 100, LCDR2 having sequence number A7 (in Figure 1), and LCDR3 having sequence number 31, wherein the VL optionally includes sequence number 146 or 267. (n) A VH comprising HCDR1 having sequence number 78, HCDR2 having sequence number 55, and HCDR3 having sequence number 9, wherein the VH may optionally be the same as the VH comprising sequence number 124. A VL including LCDR1 having sequence number 101, LCDR2 having sequence number A8 (in Figure 1), and LCDR3 having sequence number 32, wherein the VL optionally includes sequence number 147 or 268. (o) A VH comprising HCDR1 having sequence number 80, HCDR2 having sequence number 57, and HCDR3 having sequence number 11, wherein the VH may optionally be the same as the VH comprising sequence number 126, A VL including LCDR1 having sequence number 103, LCDR2 having sequence number A10 (in Figure 1), and LCDR3 having sequence number 34, wherein the VL optionally includes sequence number 149 or 270. (p) A VH comprising HCDR1 having sequence number 81, HCDR2 having sequence number 58, and HCDR3 having sequence number 12, wherein the VH may optionally be the same as the VH having sequence number 127. A VL including LCDR1 having sequence number 104, LCDR2 having sequence number A11 (in Figure 1), and LCDR3 having sequence number 35, wherein the VL optionally includes sequence number 150 or 271. (q) A VH comprising HCDR1 having sequence number 82, HCDR2 having sequence number 59, and HCDR3 having sequence number 13, wherein the VH may optionally be the same as the VH comprising sequence number 128. A VL including LCDR1 having sequence number 105, LCDR2 having sequence number A12 (in Figure 1), and LCDR3 having sequence number 36, wherein the VL optionally includes sequence number 151 or 272. (r) A VH comprising HCDR1 having sequence number 85, HCDR2 having sequence number 62, and HCDR3 having sequence number 16, wherein the VH may optionally be the same as the VH comprising sequence number 131. A VL including LCDR1 having sequence number 108, LCDR2 having sequence number A15 (in Figure 1), and LCDR3 having sequence number 39, wherein the VL optionally includes sequence number 154 or 275. (s) A VH comprising HCDR1 having sequence number 87, HCDR2 having sequence number 64, and HCDR3 having sequence number 18, wherein the VH may optionally be the same as the VH comprising sequence number 133, A VL including LCDR1 having sequence number 110, LCDR2 having sequence number A17 (in Figure 1), and LCDR3 having sequence number 41, wherein the VL optionally includes sequence number 156 or 277. (t) A VH comprising HCDR1 having sequence number 89, HCDR2 having sequence number 66, and HCDR3 having sequence number 20, wherein the VH may optionally be the same as the VH comprising sequence number 135. A VL including LCDR1 having sequence number 112, LCDR2 having sequence number A19 (in Figure 1), and LCDR3 having sequence number 43, wherein the VL optionally includes sequence number 158 or 279. (u) A VH comprising HCDR1 having sequence number 90, HCDR2 having sequence number 67, and HCDR3 having sequence number 21, wherein the VH may optionally be the same as the VH comprising sequence number 136, A VL including LCDR1 having sequence number 113, LCDR2 having sequence number A20 (in Figure 1), and LCDR3 having sequence number 44, wherein the VL optionally includes sequence number 159 or 280. (v) A VH comprising HCDR1 having sequence number 91, HCDR2 having sequence number 68, and HCDR3 having sequence number 22, wherein the VH may optionally be the same as the VH having sequence number 137, A VL including LCDR1 having sequence number 114, LCDR2 having sequence number A21 (in Figure 1), and LCDR3 having sequence number 45, wherein the VL optionally includes sequence number 160 or 281. and / or, (w) A VH comprising HCDR1 having sequence number 93, HCDR2 having sequence number 70, and HCDR3 having sequence number 24, wherein the VH may optionally be the same as the VH comprising sequence number 139, A VL comprising LCDR1 having sequence number 116, LCDR2 having sequence A23 (in Figure 1), and LCDR3 having sequence number 47, wherein optionally the VL may contain one or more of the VLs, including sequence number 162 or 283, is an anti-gamma variable 4 (Vγ4) antibody or a fragment thereof. An ex vivo method for modulating Vγ4T cells, comprising administering a cell population containing Vγ4T cells.

13. An ex vivo method for modifying Vγ4 T cells, comprising administering an anti-gamma variable 4 (Vγ4) antibody or a fragment thereof, which contains an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 163 to 185, to a cell population containing Vγ4 T cells.

14. An anti-gamma variable 4 (Vγ4) antibody containing an amino acid sequence having at least 80% sequence identity with one of sequence numbers 233-255 is administered to a cell population including Vγ4 T cells. An ex vivo method for regulating Vγ4 T cells, including the action of [doing something].

15. Ex. The procedure for modulating Vγ4 T cells involves administering an anti-gamma variable 4 (Vγ4) antibody or a fragment thereof, which contains a heavy chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 284-306 and / or a light chain amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 307-329, to a cell population containing Vγ4 T cells. vivo method.

16. The anti-Vγ4 antibody or a fragment thereof (i) scFv or full-length antibody, and / or (ii) The method according to any one of claims 1 to 15, wherein the antibody is a human antibody or a fragment thereof.

17. The method according to any one of claims 1 to 16, wherein the regulation includes proliferation of Vγ4 T cells.

18. The method according to claim 17, wherein the method provides a proliferated Vγ4T cell population containing more than 60% Vγ4T cells, such as more than 70% Vγ4T cells.

19. The method according to any one of claims 1 to 18, comprising culturing the cell population for at least 5 days.

20. The method according to any one of claims 1 to 19, comprising culturing the cell population in the presence of IL-2 and / or IL-15.

21. The cell population is obtained from a hematopoietic sample or a fraction thereof, according to the method according to any one of claims 1 to 20.

22. The method according to claim 21, wherein the hematopoietic sample consists of peripheral blood mononuclear cells (PBMCs) or low-density mononuclear cells (LDMCs).

23. The method according to any one of claims 1 to 20, wherein the cell population is obtained from a non-hematopoietic tissue sample such as skin, colon, intestine, mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina or other skin, mucous membrane or serous membrane samples.

24. The method according to any one of claims 1 to 23, wherein the cell population is obtained from human or non-human animal tissue.

25. A population of Vγ4T cells obtained by the ex vivo method described in any one of claims 1 to 24.

26. A composition comprising the Vγ4T cell population according to claim 25.

27. A pharmaceutical composition comprising the Vγ4 T cell population described in claim 25 together with a pharmaceutically acceptable diluent or carrier.

28. A pharmaceutical composition according to claim 27, for use as a pharmaceutical agent.

29. The pharmaceutical composition according to claim 27, for use in the treatment of cancer, infectious diseases, or inflammatory diseases.

30. A therapeutically effective amount of the Vγ4T cell population or as described in claim 25 A method for treating a cancer, infection, or inflammatory disease of a subject requiring the use of the pharmaceutical composition described in claim 27.

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