Modified gamma delta T cells

Abstract

This technology relates to methods for activating and expanding multiple subsets of gamma delta T cells. These subsets of gamma delta T cells may be modified and may contain one or more chimeric antigen receptors. The chimeric antigen receptors may contain one or more antigen-binding domains having binding specificity to TROP2 and / or PD-L1. These subsets of gamma delta T cells may express IL-15. These subsets of gamma delta T cells may contain one or more genomic modifications. These subsets of gamma delta T cells may contain TGFβR2 knockout. This technology also relates, in part, to single-domain antibodies (nanobodies) targeting TROP2, PD-L1, or TROP2 and PD-L1, their use in immunocytotechnology, their pharmaceutical compositions, their diagnostic and non-diagnostic reagents, and therapeutic and non-therapeutic methods of their use.

Description

[Technical Field]

[0001] field This technology relates, in part, to methods for activating and expanding multiple subsets of gamma delta T cells. In some embodiments, this technology relates to multiple subsets of gamma delta T cells. In some embodiments, this technology relates to activated and expanded subsets of gamma delta T cells produced by the methods described herein. In some embodiments, multiple subsets of gamma delta T cells are modified. In some embodiments, multiple subsets of gamma delta T cells include one or more chimeric antigen receptors. In some embodiments, the chimeric antigen receptor includes one or more antigen-binding domains having binding specificity to TROP2 and / or PD-L1. In some embodiments, multiple subsets of gamma delta T cells express IL-15. In some embodiments, multiple subsets of gamma delta T cells include one or more genomic modifications. In some embodiments, multiple subsets of gamma delta T cells include TGFβR2 knockout. This technology also relates, in part, to single-domain antibodies (nanobodies) targeting TROP2, PD-L1, or both TROP2 and PD-L1, their use in immunocytoengineering, their pharmaceutical compositions, their diagnostic and non-diagnostic reagents, and therapeutic and non-therapeutic methods of their use. [Background technology]

[0002] background Solid tumors present a high level of unmet medical need for the development of new, safe, effective, and available therapies. For example, pancreatic cancer is one of the deadliest malignant neoplasms globally and has the fourth highest mortality rate in the United States for all races and ethnicities, both male and female. Despite recent advances, the 5-year survival rate in the United States remains at 12.5%. The effectiveness of conventional therapies such as chemotherapy and radiation is very limited. Novel treatment options, such as the immune checkpoint inhibitor (ICI) pembrolizumab, approved by the FDA for the treatment of advanced solid tumors with high microsatellite instability, have very limited applicability in pancreatic cancer, where the prevalence of cases with high microsatellite instability is very low (0.8%). ICIs in combination with chemotherapy have not resulted in significant improvements in response and survival.

[0003] Gamma delta T (γδT) cells play a crucial role in immune surveillance against tumors and pathogen-infected cells. They are a subset of T cells with distinct characteristics and functions compared to conventional αβT cells. The γδT cell receptor (TCR) can recognize and respond to a diverse range of antigens, including stress-inducible molecules and non-peptide antigens presented on tumor cells. One key advantage of γδT cells is their ability to directly recognize and target tumor cells without requiring MHC presentation of antigens. This makes γδT cells particularly effective over αβT cells in highly invasive and metastatic tumors where the expression of MHC I, a common immune evasion mechanism, is lost, and antigen presentation and recognition by αβT cells are impaired. Furthermore, γδT cells may be a safe therapeutic option as they do not cause graft-versus-host disease.

[0004] In addition to activation via diverse γδTCRs, γδT cells are also expressed on NK cells and respond to a wide range of ligands expressed on tumor cells via activating receptors (NKG2D and DNAM-1) and innate cytotoxic receptors (NKp30, NKp44, and NKp46), further enhancing the breadth and amplitude of γδT cell antitumor activity against heterogeneous tumor populations and antigen evasion after immunotherapy.

[0005] γδ T cells can often directly exhibit potent cytotoxic activity against tumor cells without requiring prior priming and expansion, leading to rapid initiation of tumor death through the release of cytolytic molecules perforin and granzyme, as well as the expression of cell death receptor ligands FASL and TRAIL, which induce apoptosis in tumor cells. γδ T cells also possess a wide range of functions to activate other immune cells, including the secretion of pro-inflammatory cytokines IFN-γ and TNF-α to activate αβ T cells and dendritic cells (DCs), the activation of NK cells via 41BBL-41BB interactions, and the expression of CCR5 to activate antibody production and class switching in B cells. Vγ9Vδ2 T cells are also potent in antigen cross-presentation, efficiently stimulating the activation and proliferation of αβ T cells. Vγ9Vδ2 T cells also express CD16 / IgG FcgRIII to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

[0006] Of the two major subtypes of γδ T cells, Vδ1 T cells are naturally tissue-homing, as they are often found in epithelial tissue, which is the location of solid tumors. While Vγ9Vδ2 T cells are mostly found in circulation, they also infiltrate tumor tissue via chemotaxis and activation induced by a dysregulated mevalonate pathway, which leads to the accumulation of isopentenyl diphosphate (IPP) in TME. Consistently, tumor-infiltrating γδ T cells have been identified as the most favorable prognostic marker in a whole cancer study of 5,782 tumor samples across 25 different cancer types, including 3,238 tumor samples from 14 solid non-brain tumors. Further independent studies on individual tumor types using transcriptome, flow cytometry, and / or immunohistochemical analysis of tumor-infiltrating lymphocytes have confirmed that γδ T cells (as in CRC, HCC, gastric cancer, and head and neck squamous cell carcinoma), Vδ1 T cells (in NSCLC and TNBC), or Vγ9Vδ2 T cells (in bladder cancer) are positively associated with disease-free survival, overall survival, or early tumor stage in different solid tumors. Therefore, while both tissue-resident Vδ1 and circulating Vδ2 T cells can effectively infiltrate solid tumors, their effects may differ across cancer types, given the heterogeneity of the tumor microenvironment (TME) and immune responses across different cancer types. Multiple therapeutic subsets of γδ T cells (also known as polyclonal γδ T cells) would offer the advantage of tissue-independent treatment in the treatment of a wide range of solid tumor types.

[0007] Solid tumors are generally highly heterogeneous and branch early. Single target-specific treatments can lead to clone-specific negative selection, and for this reason, tumors have evolved numerous mechanisms to negate therapeutic efficacy, including antigen evasion through shedding, downregulation, or silencing of antigen expression, and immune evasion through MHC downregulation or loss of HLA heterozygosity. Furthermore, tumor-associated antigens (TAAs) are often expressed in both solid tumors and normal tissues, increasing the risk of on-target off-tumor toxicity and leading to dose-limiting toxicities for many antibody-based therapies. Finally, the highly immunosuppressive tumor microenvironment (TME) also presents significant challenges in maintaining therapeutic activity.

[0008] Monoclonal antibodies are called magic bullet drugs due to their intrinsic specificity. Therefore, monoclonal antibodies have a wide range of therapeutic applications. They can be used alone or as components for the direct delivery of drugs, toxins, or radioactive materials to cancer cells. Bispecific antibody-engaged (BiTE) antibodies and immune checkpoint-blocking (ICB) antibodies demonstrate how antibody activity and the immune system work together to produce successful targeted cancer therapies. Cancer cell-targeting antibodies can also be engineered as part of chimeric molecules (e.g., chimeric antigen receptors (CARs)) incorporated into specific cytotoxic immune cells to activate such immune cells for target-specific killing. The specificity of CAR-mediated T cell recognition is defined by the antibody domain and is independent of MHC presentation, allowing the antibody to be extended to any available target.

[0009] Chimeric antigen receptors (CARs) have a modular design with four main components: an antigen-binding domain, a hinge, a transmembrane domain, and an intracellular signaling domain. One component of a CAR is the extracellular antibody domain, which influences the specificity and potency of the engineered immune cells. The antigen-binding domain of a CAR typically consists of a variable heavy chain (VH) and a variable light chain (VL) of a monoclonal antibody, linked by a flexible linker to form a single-strand variable fragment (scFv). CARs can also be engineered to contain smaller single-domain antibodies (nanobodies) containing the VH domain of a camel heavy chain antibody, also called the variable heavy chain domain (VHH). Compared to conventional ScFV antibody fragments for CAR construction, single-domain antibodies not only offer comparable binding affinity but also provide greater flexibility in genetic engineering due to their smaller size. High binding capacity, solubility, stability, smaller size, simpler humanization, and lower immunogenicity indicate the potential of VHH domain antibodies in the construction of multispecific CARs for application to heterogeneous solid tumors.

[0010] In some cases, single-target specific treatment of solid tumors results in clone-specific negative selection or immune evasion through upcontrol of immunological checkpoints. Immunological checkpoints are a group of inhibitory and stimulating pathways that affect immune cell activity. Immune checkpoint blockers (ICBs) targeting immunosuppressive receptors such as CTLA-4, PD-1, and PD-L1 are the most widely used immunotherapeutic agents and have completely transformed cancer immunotherapy. Programmed death 1 ligand 1 (PD-L1), also known as B7-H1 or CD274, is a member of the B7 family and a ligand for PD-1. PD-L1 has a negative modulatory effect on the immune response. Binding of PD-L1 expressed by tumor cells to PD-1 can inhibit the function of immune cells and lead to immune evasion of the tumor. Certain PD-L1 inhibitors are approved to treat several solid tumors, including NSCLC, HNSCC, melanoma, and MCC. However, only a subset of patients (20-40%) benefit from this treatment, highlighting the need for improved treatment.

[0011] As a tumor-associated antigen (TAA), PD-L1 is variably expressed across various tumor types, including esophageal, gastrointestinal, pancreatic, breast, lung, and renal cancers. In many cases, PD-L1 expression is strongly upregulated in response to pro-inflammatory cytokines such as INFγ and TNFα released from activated immune cells, such as adoptively transferred CARγδ T cells. Upregulated PD-L1 on tumor cells can lead to T cell inactivation. Furthermore, TGF-β enriched with TME can also increase PD-L1 expression on tumor-associated macrophages (TAMs) as a mechanism for tumor immune evasion. Therefore, the development of CAR-T therapies that dual-target PD-L1 and other TAAs can minimize not only tumor antigen evasion but also tumor immune evasion in order to improve the efficacy of CAR-T therapy.

[0012] Trophoblast surface antigen 2 (TROP2), also known as tumor-associated calcium signaling molecule 2, is a type I membrane glycoprotein that is significantly overexpressed in various solid tumors. A comprehensive analysis of 18,563 tumor samples from 150 different tumor (sub)types identified TROP2 positivity in over 100 different tumor (sub)types. Of the 86 epithelial tumor entities, 97.7% showed detectable TROP2 expression, and 90.7% showed at least one case with strong positivity. Furthermore, high TROP2 expression was associated with disease progression in multiple cancer types. A meta-analysis found that TROP2 overexpression was significantly associated with poor survival (overall survival (OS) or disease-free survival (DFS)) in human solid tumors, indicating that TROP2 is a useful prognostic biomarker and a potential target for cancer therapy. New evidence suggests that TROP2-targeted therapy is effective and safe in patients with multiple prior treatments.

[0013] TROP2 can be used as a target for antibody-drug conjugate (ADC) therapy. In one example, anti-TROP2 ADC can be used for the treatment of locally advanced or metastatic unresectable triple-negative breast cancer (TNBC), urothelial cancer, and hormone receptor-positive and HER2-negative breast cancer. Another TROP2-targeted ADC has shown promising treatment results in advanced non-small cell lung cancer (NSCLC). Therefore, ADCs targeting TROP2 may be particularly useful for the treatment of metastatic triple-negative breast cancer, urothelial cancer, and NSCLC. However, excessive toxicity, increased tumor resistance to ADC drug payloads, and low response rates of ADCs pose ongoing challenges. Safer and more effective strategies are needed for TROP2-targeted treatments. Immune cells engineered with anti-TROP2 antibody binders may be a useful treatment approach for TROP2-positive cancers.

[0014] Multiple subsets of genetically engineered γδT cells are provided herein that may be useful as a safe, effective, and available treatment to address the unmet high medical needs in patients with a wide range of solid tumors. Specifically, the modified γδT cells described herein are multiple subsets derived from healthy donors and include γδT cells expressing a bispecific CAR that targets trophoblast cell surface antigen 2 (TROP2) and PD-L1, where these targets are either widely expressed on epithelial tumors (TROP2) or upregulated in response to pro-inflammatory cytokines (PD-L1). PD-L1 is an immune checkpoint factor and tumor-associated antigen. Thus, targeting PD-L1 not only minimizes tumor antigens and immune evasion, but also avoids PD-L1-mediated immunosuppression and maintains the activity of the modified γδT cells described herein. Depleting PD-L1-expressing tumor cells, stromal cells, and immune cells in the TME also disrupts the structural integrity of the fibrotic stroma, enhances lymphocyte infiltration, thereby remodeling the TME into an immune-inflammatory state.

[0015] The modified γδ T cells described herein also express IL-15, an immunostimulatory cytokine that enhances the activity, survival, and proliferative capacity of γδ T cells and innate immune cells ex vivo and in vivo. Further, in the modified γδ T cells described herein, TGFβR2 is knocked out (i.e., the TGFβR2 gene is edited by CRISRP / Cas9), thereby overcoming the immunosuppressive characteristics of the tumor microenvironment (TME), which are prominent in solid tumors, particularly pancreatic cancer and other epithelial tissue cancers. Summary of the Invention Means for Solving the Problems

[0016] Abstract In certain embodiments, provided is a method of selectively expanding gamma-delta T cells ex vivo, comprising expanding the number of gamma-delta T cells in a source cell population under expansion conditions, thereby generating an expanded gamma-delta T cell-enriched cell population, wherein the expansion conditions include a binder selected from two or more of (i) a Cluster of Differentiation 3 (CD3) binder, (ii) a Cluster of Differentiation 2 (CD2) binder, (iii) an NKp46 binder, (iv) an NKp44 binder, and (v) an NKp30 binder.

[0017] In certain embodiments, provided is a kit for selectively expanding gamma-delta T cells ex vivo, comprising a binder selected from two or more of (i) a Cluster of Differentiation 3 (CD3) binder, (ii) a Cluster of Differentiation 2 (CD2) binder, (iii) an NKp46 binder, (iv) an NKp44 binder, and (v) an NKp30 binder.

[0018] In certain embodiments, provided are modified gamma-delta T cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a first antigen-binding domain having binding specificity for TROP2 and a second antigen-binding domain having binding specificity for PD-L1.

[0019] In certain embodiments, nucleic acids encoding a chimeric antigen receptor (CAR) are also provided, the nucleic acid comprising (i) a first polynucleotide encoding a first antigen-binding domain having binding specificity to TROP2, and (ii) a second polynucleotide encoding a second antigen-binding domain having binding specificity to PD-L1.

[0020] In certain embodiments, a chimeric antigen receptor (CAR) is also provided, comprising a first antigen-binding domain having binding specificity to TROP2 and a second antigen-binding domain having binding specificity to PD-L1.

[0021] In certain embodiments, pharmaceutical compositions comprising the modified gamma delta T cells described herein are also provided.

[0022] In addition, in certain embodiments, a treatment method is also provided which includes administering a therapeutic dose of the modified gamma delta T cells described herein.

[0023] Furthermore, a) a hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86 and 90; b) SEQ ID NOs. 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, sequence number Also provided herein are agents conjugated to TROP2 or a fragment thereof, comprising a hypervariable region H2 (HVR-H2) polypeptide selected from number 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87 and SEQ ID NO: 91; and c) a hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88 and SEQ ID NO: 92.

[0024] Also provided herein are agents that bind to PD-L1 or a fragment thereof, comprising a) a hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 2, SEQ ID NOs. 6 and SEQ ID NOs. 10; b) a hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 3, SEQ ID NOs. 7 and SEQ ID NOs. 11; and c) a hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs. 4, SEQ ID NOs. 8 and SEQ ID NOs. 12.

[0025] Specific implementations are further described in the following description, examples, claims, and drawings. [Brief explanation of the drawing]

[0026] The drawings illustrate, but are not limited to, a specific implementation of this technology. For clarity and ease of explanation, the drawings are not made to scale, and in some cases, various implementations may be exaggerated or enlarged to facilitate understanding of a particular implementation.

[0027] [Figure 1A-1] Figures 1A, 1B, 1C, 1D, and 1E show the composition, expansion, and activity of gamma delta (γδ) T cells activated and expanded using anti-CD3 antibodies with or without anti-CD2 antibodies. [Figure 1A-2] Same as above. [Figure 1B-1] Same as above. [Figure 1B-2] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above.

[0028] [Figure 2] Figures 2A, 2B, and 2C show the purity of expanded γδT cells, the efficiency of TROP2-PD-L1 CAR retroviral vector transduction, and TGFβR2 gRNA / Cas9-mediated gene knockout. NT: Unmodified γδT cells; CAR: TROP2-PD-L1 CAR-IL-15 γδT cells; PLB-001: TROP2-PD-L1 CAR-IL-15, TGFβR2 KO γδT cells. The bar graphs represent results from two healthy donors (mean + / - SD).

[0029] [Figure 3]Figure 3 shows the memory phenotypes determined by CD62L and CD45RA in flow cytometry. TCM: Central memory; TNAIVE: Naive; TEM: Effector memory; TEMRA: Terminally differentiated effector memory T cells. Activation markers: CD69+ or NKG2D+; Exhaustion markers: PD1+TIGIT+ or PD1+LAG3+. NT: Unmodified γδ T cells; CAR: TROP2-PD-L1 CAR-IL-15 γδ T cells; PLB-001: TROP2-PD-L1 CAR-IL-15, TGFβR2 KO γδ T cells. The bar graph represents results (mean + / -SD) from two healthy donors.

[0030] [Figure 4] Figure 4 shows the CAR-dependent and independent innate cytotoxicity of γδT cells co-cultured with GFP-Luc-labeled tumor cell lines HCC1806 (TNBC), NCI-H1975 (NSCLC), BXPC3 (PDAC), and K562 (CML) at variable E:T ratios from 1:9 to 9:1. Tumor-specific lysis was measured by luciferase assay. NT: Unmodified γδT cells; CAR: TROP2-PD-L1 CAR-IL-15 γδT cells;

[0031] [Figure 5] Figure 5 shows the enhanced tumor-killing activity of PLB-001 against a wide range of tumor types. GFP-Luc-expressing tumor cell lines were co-cultured with specific γδT cells from two healthy donors in a 1:2 E:T ratio for up to 5 days. Fluorescent cell images were acquired on day 4 using BIOTEK CYTATION 5 (Agilent), and luciferase assays were performed on day 5. Tumor luciferase activity percentages were calculated compared to tumors alone. NT: unmodified γδT cells; CAR: TROP2-PD-L1 CAR-IL-15 γδT cells; PLB-001: TROP2-PD-L1 CAR-IL-15, TGFβR2 KO γδT cells. Statistical analysis was performed using GRAPHPAD PRISM 9 with paired t-tests. *P<0.05, **P<0.01.

[0032] [Figure 6-1] Figure 6 shows that TGFβR2 knockout maintains the tumor-killing activity of PLB-001 in the presence of TGFβ1. GFP-Luc-expressing tumor cell lines BXPC3, HPAFII, NCI-H1975, or HCC1806 were co-cultured with specific γδ T cells derived from two healthy donors in an E:T ratio of 1:1 (Panel A) or 1:2 (Panels B, C, D). Tumor-specific lysis was measured by luciferase assay. Levels of IFNγ, perforin, and granzyme B in the culture medium were measured by ELISA. Statistical analysis was performed using GRAPHPAD PRISM 9 with multiple paired t-tests. *P<0.05, **P<0.01. NT: Unmodified γδT cells; CAR: TROP2-PD-L1 CAR-IL-15 γδT cells; PLB-001: TROP2-PD-L1 CAR-IL-15, TGFβR2 KO γδT cells. [Figure 6-2] Same as above.

[0033] [Figure 7] Figure 7 shows, rows A and B, the effective tumor invasion and killing by PLB-001 in a 3D spheroid model derived from pancreatic tumor cell lines. 5,000 GFP-Luc-expressing BxPC3 and HPAF-II cells were seeded into tumor spheroids and cultured for 3 days. Then, 15,000 (A) or 5,000 (B) NT or CARγδT cells or PLB-001 cells obtained from two healthy donors were added. The data shown are mean + / - SD (n=4), and statistical analysis was performed using GRAPHPAD PRISM 9 with paired t-tests. *P<0.05, **P<0.01. NT: unmodified γδT cells; CAR: TROP2-PD-L1 CAR-IL-15 γδT cells; PLB-001: TROP2-PD-L1 CAR-IL-15, TGFβR2 KOγδT cells.

[0034] [Figure 8] Figure 8 shows an example of a chimeric antigen receptor (CAR) construct.

[0035] [Figure 9]Figure 9 shows the transduction efficiency of the PD-L1 CAR retroviral vector, as evaluated by flow cytometry, in expanded γδT cells derived from two healthy donors.

[0036] [Figure 10] Figure 10 shows the expression of the degranulation marker CD107a (upper panel) and IFN-γ (lower panel) in PD-L1 CARγδT cells co-cultured with tumor cells.

[0037] [Figure 11A] Figures 11A, 11B, and 11C show the antitumor cytotoxic effects of PD-L1 CARγδT cells in the ovarian cancer cell line SKOV3 (Figure 11A) and the lung cancer cell lines HCC827 (Figure 11B) and NCI-H1975 (Figure 11C). [Figure 11B] Same as above. [Figure 11C] Same as above.

[0038] [Figure 12A] Figures 12A and 12B show the antitumor activity of PD-L1 CARγδT cells in 3D tumor spheroids derived from GFP-Luc-labeled lung cancer cell lines HCC827 (Figure 12A) and NCI-H1975 (Figure 12B). [Figure 12B] Same as above.

[0039] [Figure 13A] Figures 13A and 13B show the identification of lead phage clone conjugates that specifically bind to TROP2-expressing cell lines. [Figure 13B] Same as above.

[0040] [Figure 14] This study demonstrates efficient transduction of γδT cells using the TROP2 CAR retroviral vector. Cell surface CAR expression was detected using QBEND / 10, an antibody specific to the CD34 tag in the CAR construct.

[0041] [Figure 15A] Figures 15A, 15B, and 15C show the antigen-binding ability of TROP2 CARγδT cells as determined by flow cytometry. Binding of recombinant human TROP2 protein to the indicated CAR binder on γδT cells was detected by anti-human Fc antibody. [Figure 15B] Same as above. [Figure 15C] Same as above.

[0042] [Figure 16] Figure 16 shows the EC50 of the TROP2 protein in TROP2-binding-expressing γδT cells.

[0043] [Figure 17A] Figures 17A, 17B, 17C, and 17D show the antitumor cytotoxicity of TROP2 CARγδT cells co-cultured with breast cancer cell line HCC1806, lung cancer cell line NCI-H1975, pancreatic cancer cell line HPAF-II, and gastric cancer cell line N87. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above.

[0044] [Figure 18A] Figures 18A and 18B show the tumor invasion and killing capacity of TROP2 CARγδT cells in tumor spheroids derived from HPAF-II and NCI-H1975 cell lines. [Figure 18B] Same as above.

[0045] [Figure 19A] Figures 19A, 19B, and 19C show the sequential killing ability of TROP2 CARγδT cells during repeated challenges of tumor cell lines NCI-H1975, N87, and HPAF-II. [Figure 19B] Same as above. [Figure 19C] Same as above.

[0046] [Figure 20A]Figure 20A shows the transduction efficiency of the TROP2-PD-L1 CAR retroviral vector. [Figure 20B] Figure 20B shows the composition of enlarged TROP2-PD-L1 CARγδT cells.

[0047] [Figure 21A] Figures 21A, 21B, and 21C show the antitumor cytotoxicity of TROP2-PD-L1 CARγδT cells co-cultured with lung cancer cell lines NCI-H1975 and HCC827, and ovarian cancer cell line SKOV3. [Figure 21B] Same as above. [Figure 21C] Same as above.

[0048] [Figure 22A] Figures 22A, 2B, and 22C show the antitumor activity of TROP2-PD-L1 CARγδT cells in tumor spheroids derived from the NCI-H1975 cell line. [Figure 22B] Same as above. [Figure 22C] Same as above.

[0049] [Figure 23-1] Figures 23A, 23B, 23C, and 23D show equivalent cell viability, expansion, and composition of multiple subsets of γδT cells expanded with either IL-2 or IL-15. [Figure 23-2] Same as above.

[0050] [Figure 24-1] Figures 24A, 24B, 24C, and 24D show the efficiency of TGFβR2 knockout by electroporation of TGFβR2 gRNA / Cas9 RNPs in expanding γδT cells, as measured by flow cytometry of surface TGFβR2 expression. The viability and expansion capacity of γδT cells after RNP electroporation were also evaluated. [Figure 24-2] Same as above.

[0051] [Figure 25A] Figures 25A, 25B, 25C, 25D, and 25E show the serial killing activity of TGFβR2 knockout, TROP2-PD-L1 CAR-IL-15γδT cells (PLB-001) during repeated challenges with tumor cell lines NCI-H1975, HPAFII, or BXPC3. Proliferation of PLB-001 and γδT cell subsets, as well as exhaustion markers after repeated exposure to tumor cells, were also determined by flow cytometry. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above.

[0052] [Figure 26-1] Figures 26A, 26B, 26C, 26D, and 26E show the antitumor activity of PLB-001 after intravenous administration in a subcutaneous mammary tumor xenograft model of NSG mice. Tissue invasion and tumor-inducible expansion of PLB-001 were determined by analyzing tumor and normal tissue. [Figure 26-2] Same as above. [Modes for carrying out the invention]

[0053] Detailed explanation Methods for ex vivo activation and selective expansion of gamma delta T cells are provided herein. Populations of activated and expanded gamma delta T cells are also provided herein. In certain embodiments, gamma delta T cells are modified. For example, the modification may include the introduction of a chimeric antigen receptor (CAR) and / or a genomic modification. The CAR may include one or more antigen-binding domains (e.g., TROP2 antigen-binding domain; PD-L1 antigen-binding domain). The CAR may include one or more cytokines (e.g., IL-15). The genomic modification may include a specific gene knockout (e.g., TGFβR2 knockout).

[0054] cell culture Methods and compositions for cell culture are provided herein. In particular, extended culture conditions ("extended conditions") are provided herein. Cell culture or culture typically refers to the maintenance of cells in an artificial in vitro environment or in an external ex vivo environment (i.e., outside of an organism). The specific cell culture systems described herein may be ex vivo and / or in vitro environments.

[0055] Cells can be obtained from a subject and / or a cell source. Cells obtained from a subject and / or a cell source may be called the origin cell population. The origin cell population is the input population of cells for expansion under the culture conditions (e.g., expansion conditions) described herein. The cell source may include a population of circulating blood cells. The cell source may include a population of peripheral blood mononuclear cells (PBMCs). The cell source may include a population of immune cells. The cell source may include a population of T cells. The cell source may include a population of alpha-beta T cell-depleted T cells. The origin cell population can be obtained from a subject in various ways (e.g., isolated from circulation, isolated by apheresis, isolated by leukocyte apheresis). In some embodiments, immune cells are isolated from the subject. In some embodiments, lymphocytes are isolated from the subject. In some embodiments, T cells are isolated from the subject. In some embodiments, cells may be derived from (e.g., obtained from) a population of peripheral blood mononuclear cells (PBMCs) isolated from the subject. The subjects may include any mammal, such as mice, rats, dogs, cats, cattle, horses, pigs, non-human primates, and humans, but may not be limited to any other animal. In some embodiments, the subjects are humans.

[0056] The cell seeding density can be adjusted according to specific desired culture conditions. For example, 1 cm 2 Approximately 1 x 10 3 ~About 1~10×10 5 An initial seeding density of 1 cm can be used. In some embodiments, 1 cm 2 Approximately 1-10-10x10 5The initial seeding density of a single cell can be used. In a particular case, 1 × 10⁻⁶ 6 Each cell is 75 cm 2 Cells may be cultured in culture flasks. In certain cases, cell culture bags are used. In certain cases, membrane-based static cell culture systems are used (e.g., G-REX by SCALEREADY). Cell density may be changed as needed at any passage.

[0057] Cells can be cultured in a cell incubator at approximately 37°C under normal atmospheric pressure. The incubator atmosphere may be humidified and may contain approximately 3–10% carbon dioxide in the air. In some cases, the incubator atmosphere may contain approximately 0.1–30% oxygen. Temperature, pressure, and carbon dioxide and oxygen concentrations can be changed as needed. The pH of the culture medium may be in the range of approximately 7.1–7.6, or approximately 7.1–7.4, or approximately 7.1–7.3.

[0058] Cell culture media may be changed as needed, at intervals of 1-2 days or more or less. As cells approach confluence in the culture vessel, they may be passaged. Cell passage is the process of dividing or dividing cells and transferring some of the cells to a new culture vessel or culture environment. Cells adhering to the cell culture surface may require detachment. Methods for detaching adherent cells from the culture vessel surface are well known and may include the use of enzymes such as trypsin.

[0059] A single passage refers to a single division or manual division of cells and the transfer of a smaller number of cells to a new container or environment. When passaged, cells can be divided into any ratio that allows the cells to adhere and grow. For example, in a single passage, cells can be divided into a ratio of 1:2, 1:3, 1:4, 1:5, etc. In some embodiments, cells are passaged at least about 1 to at least about 300 times. For example, cells can be passaged at least about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 times. In some embodiments, cells are passaged at least about 15 times. In some embodiments, cells are passaged at least about 25 times.

[0060] Cell proliferation generally refers to cell division in which one mother cell divides into two daughter cells. Cell proliferation may also be called cell expansion. As used herein, cell proliferation generally does not mean an increase in the actual size of the cell (e.g., diameter, volume). The stimulus for cell proliferation can be evaluated by plotting a cell population (e.g., cell population doubling) over time. Cell populations with steeper growth curves are generally considered to grow faster than cell populations with less steep curves. For example, growth curves can be compared for various treatments between the same cell type, or growth curves can be compared for different cell types under the same conditions.

[0061] The expansion of a cell population can be expressed as population doubling. When cells in culture divide so that the number of cells doubles, cell doubling occurs. In some examples, cells are counted to determine whether the cell population has doubled, tripled, or increased by other factors. The number of population doublings may not be equivalent to the number of times the cell culture is passaged. For example, passaged cells and dividing them in a 1:3 ratio for further culture may not be equivalent to tripling the cell population. A formula that may be used to calculate population doubling (PD) is presented in formula A.

number

[0062] Here, n = the final PD number of the cell culture when collected or passaged, Y = the cell yield when collected or passaged, l = the number of cells used as the inoculum to initiate the cell culture, and X = the PD number of the origin cell culture used to initiate the subculture.

[0063] In some embodiments, the methods herein include expanding a population of cells. Expanding a population of cells can be referred to as growing a population of cells. The expansion of the cell population can be expressed as a fold increase in the number of cells. An equation that can be used to calculate the fold increase as a function of population doublings is shown in Equation B.

Equation

[0064] Where F = the fold increase in the number of cells after n population doublings. For example, after 1 population doubling, the number of cells increases 2-fold, after 2 population doublings, the number of cells increases 4-fold (2 2 = 4)-fold, after 3 population doublings, the number of cells increases 8-fold (2 3 = 8)-fold, and so on. Thus, after 20 (twenty) population doublings, the number of cells increases more than 1 million-fold (2 20 = 1,048,576), after 30 (thirty) population doublings, the number of cells increases more than 1 billion-fold (2 30 = 1,073,741,824), and after 40 (forty) population doublings, the number of cells increases more than 1 trillion-fold (2 40The number of cells increases by, for example, 1,099,511,627,776. In some embodiments, the cell population can be expanded or enlarged by at least about 2 times to at least about 1 trillion times. For example, the cell population can be expanded by at least about 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 1,000 times, 10,000 times, 100,000 times, 1,0

[0065] Gamma delta T cell expansion In certain embodiments, methods and compositions for expanding a population of gamma delta T cells are provided herein. In some embodiments, methods and compositions are provided for selectively expanding a population of gamma delta T cells (i.e., without or with minimal corresponding expansion of other immune cells (e.g., alpha-beta T cells, NK cells)). Selective expansion of gamma delta T cells generates an expanded cell population enriched with gamma delta T cells.

[0066] In some embodiments, the expansion condition includes one or more agents that bind to one or more antigens. In the context of expansion condition components as used herein, the binder generally refers to one or more antibodies, antibody fragments, or antibody derivatives, or molecules containing them. In some examples, the binder may be a ligand. The term “antibody” is used herein in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. An antibody fragment generally refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab'-SH, F(ab')2, and multispecific antibodies formed from antibody fragments. An antibody derivative generally refers to a molecule other than an intact antibody that contains a portion derived from an intact antibody (or its antigen-binding fragment) and binds to the antigen to which the intact antibody (or its antigen-binding fragment) binds. Examples of antibody derivatives include, but are not limited to, aptamers containing multiple antigen-binding antibody fragments, such as single-chain variable fragments (scFv), VHH fragments, nanobodies, diabodies, and triabodies.

[0067] In some embodiments, the extensions herein include one or more agents that bind to cell surface molecules (e.g., immune cell markers). In some embodiments, the extensions herein include one or more differentiation cluster (CD) conjugates. Differentiation clusters (also known as clusters of designation or classification determinants) are generally cell surface markers used for immunophenotyping of cells. CD molecules can act as receptors or ligands, often initiating signal cascades and altering cell behavior. Some CD proteins do not play a role in cell signaling but have other functions, such as cell adhesion. Human CDs are numbered from 1 to 371 (e.g., CD1 to CD371). In some embodiments, the extensions herein include CD2 conjugates. In some embodiments, the CD2 conjugate is an antibody, antibody fragment, or antibody derivative. Any suitable anti-CD2 antibody, antibody fragment, or antibody derivative, including commercially available anti-CD2 antibodies, antibody fragments, or antibody derivatives, can be used in the extensions herein. In some embodiments, the extensions herein include CD3 conjugates. In some embodiments, the CD3 conjugate is an antibody, antibody fragment, or antibody derivative. Any suitable anti-CD3 antibody, antibody fragment, or antibody derivative, including commercially available anti-CD3 antibodies, antibody fragments, or antibody derivatives, can be used in the expanded conditions specified herein. In some embodiments, the expanded conditions include a binder comprising a CD3 binder and a CD2 binder. In some embodiments, the expanded conditions include a binder comprising a CD3 binder and a CD2 binder.

[0068] In some embodiments, the expansion condition herein includes one or more agents that bind to NKp44, NKp46, and / or NKp30. One or more agents that bind to NKp44 may include an NKp44 ligand (e.g., PDGF-DD). One or more agents that bind to NKp46 may include an anti-NKp46 antibody, a fragment thereof, or a derivative thereof. One or more agents that bind to NKp30 may include an anti-NKp30 antibody, a fragment thereof, or a derivative thereof. In some embodiments, the expansion condition includes a binder comprising a CD3 binder and an NKp44 binder. In some embodiments, the expansion condition includes a binder comprising a CD3 binder and an NKp46 binder. In some embodiments, the expansion condition includes a binder comprising a CD3 binder and an NKp46 binder. In some embodiments, the expansion condition includes a binder comprising a CD3 binder and an NKp30 binder. In some embodiments, the expansion condition includes a binder consisting of a CD3 binder and an NKp30 binder.

[0069] In some embodiments, the expanded conditions herein include one or more cytokines. Cytokines are generally small proteins (about 5–25 kDa) involved in cell signaling. Cytokines typically exert their functions by interacting with specific cytokine receptors on the surface of target cells. Cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are generally produced by immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as cells including endothelial cells, fibroblasts, and various stromal cells.

[0070] In some embodiments, the specified extension includes one or more interleukins. Interleukins (ILs) are a group of cytokines expressed and secreted by leukocytes (leukocytes) and several other somatic cells. Interleukins may include IL-1 to IL-36. The majority of interleukins are synthesized by CD4 helper T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T lymphocytes, B lymphocytes, and hematopoietic cells. In some embodiments, the specified extension includes interleukin-15 (IL-15). In some embodiments, the specified extension includes one or more cytokines consisting of IL-15. In some embodiments, the specified extension includes one or more interleukins consisting of IL-15. In some embodiments, the specified extension includes interleukin-2 (IL-2). In some embodiments, the specified extension includes one or more cytokines consisting of IL-2. In some embodiments, the specified extension includes one or more interleukins consisting of IL-2.

[0071] In some embodiments, the number of cells is multiplied several times in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. The cells multiplied by a particular magnification may refer to all cells in the cell population or to specific cells in the population (e.g., T cells, gamma delta T cells). In some embodiments, the number of cells is multiplied by about 100 times or more in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of cells is multiplied by about 200 times or more in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of cells is multiplied by about 300 times or more in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of cells is multiplied by about 400 times or more in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of cells is increased by approximately 500 times or more in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of cells is increased by approximately 200 to approximately 300 times in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population.

[0072] The cell population expanded by the method described herein may include an enriched gamma delta T cell population. For example, the cell population expanded by the method described herein may contain about 80% or more gamma delta T cells. In some embodiments, the cell population expanded by the method described herein may contain about 85% or more gamma delta T cells. In some embodiments, the cell population expanded by the method described herein may contain about 90% or more gamma delta T cells. In some embodiments, the cell population expanded by the method described herein may contain about 95% or more gamma delta T cells. In some embodiments, the cell population expanded by the method described herein may contain about 100% gamma delta T cells. In some embodiments, the cell population expanded by the method described herein may contain about 90% to 95% gamma delta T cells.

[0073] The cell population expanded by the method described herein may be depleted of non-gamma delta T cells. For example, the cell population expanded by the method described herein may be depleted of natural killer (NK) cells and / or alpha-beta T cells. In some embodiments, the expanded gamma delta T cell-enriched cell population contains about 5% or less natural killer (NK) cells. In some embodiments, the expanded gamma delta T cell-enriched cell population contains about 4% or less natural killer (NK) cells. In some embodiments, the expanded gamma delta T cell-enriched cell population contains about 3% or less natural killer (NK) cells. In some embodiments, the expanded gamma delta T cell-enriched cell population contains about 2% or less natural killer (NK) cells. In some embodiments, the expanded gamma delta T cell-enriched cell population contains about 1% or less natural killer (NK) cells. In some embodiments, the expanded gamma delta T cell-enriched cell population does not contain any detectable natural killer (NK) cells.

[0074] In some embodiments, the expanded gamma-delta T cell enriched cell population contains about 5% or less alpha-beta T cells. In some embodiments, the expanded gamma-delta T cell enriched cell population contains about 4% or less alpha-beta T cells. In some embodiments, the expanded gamma-delta T cell enriched cell population contains about 3% or less alpha-beta T cells. In some embodiments, the expanded gamma-delta T cell enriched cell population contains about 2% or less alpha-beta T cells. In some embodiments, the expanded gamma-delta T cell enriched cell population contains about 1% or less alpha-beta T cells. In some embodiments, the expanded gamma-delta T cell enriched cell population does not contain any detectable alpha-beta T cells.

[0075] The gamma delta T cell-enriched cell population expanded by the methods described herein may include one or more subpopulations of gamma delta T cells. These subpopulations may include Vdelta1 (Vδ1) T cells, Vdelta2 (Vδ2) T cells, or non-Vδ1 and Vδ2 T cells. The gamma delta T cell-enriched cell population expanded by the methods described herein may include multiple subsets of gamma delta T cells. In some embodiments, the expanded subsets of the gamma delta T cell population include subpopulations of Vdelta1 (Vδ1) T cells and Vdelta2 (Vδ2) T cells. In some embodiments, the subsets of gamma delta T cells include subpopulations of Vdelta1 (Vδ1) T cells, Vdelta2 (Vδ2) T cells, and non-Vδ1 and Vδ2 T cells.

[0076] The magnification ratio of each gamma delta T cell subpopulation may vary under the magnification conditions described herein. For example, the number of Vδ1 T cells may be magnified by about 1,000 times or more in the magnified gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells may be magnified by about 2,000 times or more in the magnified gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells may be magnified by about 3,000 times or more in the magnified gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells may be magnified by about 4,000 times or more in the magnified gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells may be magnified by about 5,000 times or more in the magnified gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells can be increased by approximately 6,000 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells can be increased by approximately 1,000 to approximately 6,000 times in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells can be increased by approximately 1,500 to approximately 2,500 times in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells can be increased by approximately 2,000 times in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ1 T cells can be increased by approximately 10,000 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population.In some embodiments, the number of Vδ1 T cells can be increased by approximately 8,000 to 12,000 times in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population.

[0077] In another example, the number of Vδ2 T cells may be increased by approximately 100 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells may be increased by approximately 200 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells may be increased by approximately 300 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells may be increased by approximately 400 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells may be increased by approximately 500 times or more in an expanded gamma-delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by about 600 times or more in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by about 700 times or more in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by about 800 times or more in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by about 900 times or more in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by about 1,000 times or more in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by approximately 100 to 1,400 times in the expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population.In some embodiments, the number of Vδ2 T cells can be increased by approximately 200 to 1,000 times in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by approximately 600 to 800 times in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by approximately 700 times in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by approximately 300 to 400 times in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population. In some embodiments, the number of Vδ2 T cells can be increased by approximately 350 times in an expanded gamma delta T cell-enriched cell population compared to the number of cells in the origin cell population.

[0078] In some embodiments, the expanded gamma delta T cell population includes varying amounts of subpopulations. The amount (e.g., percentage) may refer to a percentage of gamma delta T cells or a percentage of all cells in the population. For example, the expanded gamma delta T cell population may contain about 5–60% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 10–60% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 20–60% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 30–60% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 40–60% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 50–55% Vδ1 T cells. In some embodiments, the expanded gamma delta T cell population contains about 53–54% Vδ1 T cells.

[0079] In some embodiments, the expanded gamma delta T cell population contains approximately 25–95% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–85% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–75% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–65% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–55% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–45% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 25–35% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 28–33% Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 30–31% Vδ2 T cells.

[0080] In some embodiments, the expanded gamma delta T cell population contains approximately 5–15% non-Vδ1 & Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 6–11% non-Vδ1 & Vδ2 T cells. In some embodiments, the expanded gamma delta T cell population contains approximately 8–9% non-Vδ1 & Vδ2 T cells.

[0081] In some embodiments, the methods herein include isolating gamma delta T cells from an expanded gamma delta T cell enriched cell population. In some embodiments, the methods herein include modifying gamma delta T cells in an expanded gamma delta T cell enriched cell population. The expanded gamma delta T cells may be modified (e.g., genetically engineered) by modifications described herein (e.g., introduction of CARs, genomic modification). In some embodiments, the methods herein include storing the expanded gamma delta T cell enriched cell population or its isolates in a cell bank. In some embodiments, the gamma delta T cells described herein may provide a clinical-grade cell bank that can be used for a large number of patients. In some embodiments, the bank is appropriately populated with gamma delta T cells obtained from healthy volunteer donors (e.g., of blood type O). The collected and processed gamma delta T cells described herein may be stored for future use in a cell bank or depository. Thus, the cells may be stored in a cryoprotective agent such as DMSO or CryoStor® and subjected to controlled-rate freezing and storage in liquid nitrogen. Gamma delta T cells can be stored in unitized stocks of prescribed units or doses, as needed for one or more treatment steps.

[0082] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) are provided herein. In some embodiments, modified gamma delta T cells include CARs. A CAR generally comprises one or more polypeptide components that recognize one or more target antigens (extracellular domain; antigen-recognition domain; antigen-binding domain), which refer to a chimeric polypeptide linked to a transmembrane polypeptide and an intracellular domain polypeptide selected to activate T cells. The antigen-recognition domain may be an antibody component (e.g., a single-strand variable fragment (ScFv), an antigen-binding fragment (VHH fragment) of a heavy-chain-only antibody, a nanobody, etc.) or may be derived from other molecules, e.g., T cell receptors or pattern recognition receptors. The intracellular domain typically includes at least one polypeptide that causes T cell activation, e.g., CD3 zeta, and, for example, a costimulatory domain, e.g., CD28, OX40, CD40L, ICOS, CD27, NKG2D, DNAM-1, DAP10, DAP12, and 4-1BB, for example. CAR can also refer to chimeric receptors that are not antibody-derived but are chimeric T cell receptors. Chimeric T cell receptors may contain polypeptide sequences that recognize a target antigen, and these recognition sequences may, for example, be derived from T cell receptors or scFvs, but are not limited to these. The intracellular domain polypeptide acts to activate T cells.

[0083] The CARs described herein may comprise one or more antigen-binding domains. The antigen-binding domains may bind specifically to a target. The terms "specifically binding," "specific," and similar terms generally refer to a molecule or domain (e.g., an antigen-binding domain) that binds to a target with at least twice the affinity of a non-target compound, e.g., at least four, five, six, seven, eight, nine, ten, twenty, twenty-five, fifty, or one hundred times greater affinity. For example, an antigen-binding domain that specifically binds to a target typically binds to the target with at least twice the affinity of a non-target. Specificity can be determined using standard methods, e.g., solid-phase ELISA immunoassays. The term "binding with respect to a target" typically indicates that the antigen-binding domain binds to a large portion of the target in a pure population (assuming appropriate molar ratios). For example, an antigen-binding domain that binds to a given target typically binds to at least about two-thirds of a particular target in solution (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a particular target in solution).

[0084] In some embodiments, the CARs described herein include a VH-VL dimer or a single-chain antibody (an antibody existing as a single polypeptide chain), or a fragment thereof, such as a single-chain Fv antibody (sFv or scFv) in which a variable heavy-chain domain and a variable light-chain domain are bound together (directly or via a peptide linker) to form a continuous polypeptide. A single-chain Fv antibody is typically covalently linked VH-VL and can be expressed from nucleic acids containing VH- and VL-coding sequences that are directly linked or linked by a peptide-coding linker. Although VH and VL are linked to each other as a single polypeptide chain, the VH and VL domains typically associate non-covalently. Both the VH and VL domains typically contain six hypervariable regions (HVRs) (also called complementarity-determining regions (CDRs)) (three in the heavy chain and three in the light chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. In certain cases, a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to a target. A dsFv is an Fv having an engineered intermolecular disulfide bond that stabilizes the VH-VL pair. In some embodiments, the CARs described herein include a single-domain antibody or a fragment thereof engineered from a heavy-chain antibody (e.g., a heavy-chain antibody found in camelids), called a VHH fragment. A VHH fragment typically includes three hypervariable regions (HVRs) (also called complementarity-determining regions (CDRs)): hypervariable region H1 (HVR-H1), hypervariable region H2 (HVR-H2), and hypervariable region H3 (HVR-H3).

[0085] In some embodiments, the antigen-binding domain comprises one or more mutations (e.g., amino acid substitutions, deletions, and / or insertions). An exemplary method for identifying specific residues or regions of the antigen-binding domain that are preferred sites for amino acid substitution is alanine scanning mutagenesis. Here, a target residue or group of residues is identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction between the amino acid and the antigen. Then, amino acid sites that are functionally sensitive to the substitution are refined by introducing further or other variants at or to the substitution site. Thus, while the sites for introducing amino acid sequence mutations are predetermined, the nature of the mutations themselves does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis can be performed on the target codon or region, and the expressed antigen-binding domain variants can be screened for desired activity. Amino acid insertions can include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an N-terminal methionyl residue.

[0086] Sites for amino acid substitution may include sites within the hypervariable region and sites within the framework region. Amino acid substitutions may include conservative or non-conservative substitutions. Examples of substitutions are listed below. Ala(A):val;leu;ile Arg(R):lys;gln;asn Asn(N):gln;his;asp,lys;arg Asp(D):glu;asn Cys( C):ser;ala Gin(Q):asn;glu Glu(E):asp;gln Gly(G):ala His(H):asn;gln;lys;arg Ile(I):leu;val;met;ala;phe;norleucine Leu(L): norleucine; ile; val; met; ala; phe Lys(K):arg;gln;asn Met(M):leu;phe;ile Phe(F):leu;val;ile;ala;tyr Pro(P):ala Ser(S):thr Thr(T):ser Trp(W):tyr;phe Tyr(Y):trp;phe;thr;ser Val(V):ile;leu;met;phe;ala;norleucine

[0087] Substantial modification of the biological properties of the antigen-binding domain can be achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the region of substitution, e.g., the conformation of a sheet or helix, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulk of the side chain.

[0088] Naturally occurring residues can be grouped based on the common side-chain characteristics listed below. (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Basicity: asn, gln, his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromatics: trp, tyr, phe.

[0089] Non-conservative substitutions generally involve exchanging a member of one class for a member of another class.

[0090] In some embodiments, the CAR includes a TROP2 antigen-binding domain (e.g., anti-TROP2 VHH polypeptide; anti-TROP2 ScFv polypeptide). In some embodiments, the CAR includes a human TROP2 antigen-binding domain. In some embodiments, the CAR includes a PD-L1 antigen-binding domain (e.g., anti-PD-L1 VHH polypeptide; anti-PD-L1 ScFv polypeptide). In some embodiments, the CAR includes a human PD-L1 antigen-binding domain. In some embodiments, the CAR includes both a TROP2 antigen-binding domain (e.g., anti-TROP2 VHH polypeptide; anti-TROP2 ScFv polypeptide) and a PD-L1 antigen-binding domain (e.g., anti-PD-L1 VHH polypeptide; anti-PD-L1 ScFv polypeptide). The antigen-binding domains herein may be referred to as the first antigen-binding domain and the second antigen-binding domain, but "first" and "second" do not limit the order of the domains in the CAR. Therefore, the "first" antigen domain may appear first or second in the CAR construct, and the "second" antigen-binding domain may appear first or second in the CAR construct.

[0091] In some embodiments, the CAR includes an extracellular domain, which includes a TROP2 antigen-binding domain, a linker, a PD-L1 antigen-binding domain, and a hinge. In some embodiments, the extracellular domain includes a PD-L1 antigen-binding domain, a linker, a TROP2 antigen-binding domain, and a hinge. In some embodiments, the extracellular domain includes multiple TROP2 antigen-binding domains. In some embodiments, the extracellular domain includes multiple PD-L1 antigen-binding domains. For example, the extracellular domain may include two TROP2 antigen-binding domains and one PD-L1 antigen-binding domain. The extracellular domain may include one TROP2 antigen-binding domain and two PD-L1 antigen-binding domains. The extracellular domain may include two TROP2 antigen-binding domains and two PD-L1 antigen-binding domains. Multiple PD-L1 antigen-binding domains and / or multiple TROP2 antigen-binding domains may be present in the extracellular domain in any order.

[0092] In some embodiments, the CAR comprises a polypeptide selected from the polypeptides in Table 4 (e.g., SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107). In some embodiments, the CAR comprises a polypeptide that is at least about 80% identical to the polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107. In some embodiments, the CAR comprises a polypeptide that is at least about 85% identical to the polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107. In some embodiments, the CAR comprises a polypeptide that is at least about 90% identical to the polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107. In some embodiments, the CAR comprises a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide selected from SEQ ID NOs. 102, 103, 104, 105, 106, and 107.

[0093] In certain configurations, CAR contains an anti-PD-L1 VHH polypeptide. In certain configurations, CAR contains an anti-PD-L1 VHH polypeptide selected from the VHH polypeptides in Table 1 (e.g., SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9). In certain configurations, CAR contains an anti-PD-L1 VHH polypeptide having an amino acid sequence that is at least approximately 80% identical to the anti-PD-L1 VHH amino acid sequence in Table 1. In certain configurations, CAR contains an anti-PD-L1 VHH polypeptide having an amino acid sequence that is at least approximately 85% identical to the anti-PD-L1 VHH amino acid sequence in Table 1. In certain configurations, CAR contains an anti-PD-L1 VHH polypeptide having an amino acid sequence that is at least approximately 90% identical to the anti-PD-L1 VHH amino acid sequence in Table 1. In certain configurations, the CAR contains an anti-PD-L1 VHH polypeptide having an amino acid sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the anti-PD-L1 VHH amino acid sequences in Table 1.

[0094] In certain configurations, the CAR comprises an anti-PD-L1 VHH polypeptide containing a hypervariable region H1 (HVR-H1) selected from the HVR-H1 polypeptides of Table 1 (e.g., SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H1 polypeptide that is at least 80% identical to the HVR-H1 polypeptides of Table 1 (e.g., SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H1 polypeptide that is at least 85% identical to the HVR-H1 polypeptides of Table 1 (e.g., SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H1 polypeptide that is at least 90% identical to the HVR-H1 polypeptides of Table 1 (e.g., SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10). In some embodiments, the anti-PD-L1 VHH polypeptides provided herein contain at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H1 polypeptides to the HVR-H1 polypeptides of Table 1 (e.g., SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10).

[0095] In some embodiments, the CAR comprises an anti-PD-L1 VHH polypeptide containing a hypervariable region H1 (HVR-H1) selected from the HVR-H1 polypeptide in Table A. [Table A]

[0096] In certain configurations, the CAR comprises an anti-PD-L1 VHH polypeptide containing a hypervariable region H2 (HVR-H2) selected from the HVR-H2 polypeptides of Table 1 (e.g., SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H2 polypeptide that is at least 80% identical to the HVR-H2 polypeptides of Table 1 (e.g., SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H2 polypeptide that is at least 85% identical to the HVR-H2 polypeptides of Table 1 (e.g., SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H2 polypeptide that is at least 90% identical to the HVR-H2 polypeptides of Table 1 (e.g., SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11). In some embodiments, the anti-PD-L1 VHH polypeptides provided herein include at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H2 polypeptides to the HVR-H2 polypeptides of Table 1 (e.g., SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11).

[0097] In certain configurations, the CAR comprises an anti-PD-L1 VHH polypeptide containing a hypervariable region H3 (HVR-H3) selected from the HVR-H3 polypeptides of Table 1 (e.g., SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H3 polypeptide that is at least 80% identical to the HVR-H3 polypeptides of Table 1 (e.g., SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H3 polypeptide that is at least 85% identical to the HVR-H3 polypeptides of Table 1 (e.g., SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12). In some embodiments, the anti-PD-L1 VHH polypeptide provided herein comprises an HVR-H3 polypeptide that is at least 90% identical to the HVR-H3 polypeptides of Table 1 (e.g., SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12). In some embodiments, the anti-PD-L1 VHH polypeptides provided herein include at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H3 polypeptides to the HVR-H3 polypeptides of Table 1 (e.g., SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12).

[0098] In certain configurations, CAR contains an anti-TROP2 VHH polypeptide. In certain configurations, CAR contains an anti-TROP2 VHH polypeptide selected from the VHH polypeptides in Table 2 (e.g., SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89). In certain configurations, CAR contains an anti-TROP2 VHH polypeptide having an amino acid sequence that is at least approximately 80% identical to the anti-TROP2 VHH amino acid sequence in Table 2. In certain configurations, CAR contains an anti-TROP2 VHH polypeptide having an amino acid sequence that is at least approximately 85% identical to the anti-TROP2 VHH amino acid sequence in Table 2. In certain configurations, the CAR contains an anti-TROP2 VHH polypeptide having an amino acid sequence that is at least approximately 90% identical to the anti-TROP2 VHH amino acid sequence in Table 2. In certain configurations, the CAR contains an anti-TROP2 VHH polypeptide having an amino acid sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the anti-TROP2 VHH amino acid sequence in Table 2.

[0099] In certain configurations, the CAR contains an anti-TROP2 VHH polypeptide including a hypervariable region H1 (HVR-H1) selected from the HVR-H1 polypeptides in Table 2 (e.g., SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 80% identical HVR-H1 polypeptides to the HVR-H1 polypeptides listed in Table 2 (e.g., SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 85% identical HVR-H1 polypeptides to the HVR-H1 polypeptides listed in Table 2 (e.g., SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 90% identical HVR-H1 polypeptides to the HVR-H1 polypeptides listed in Table 2 (e.g., SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90).In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H1 polypeptides to the HVR-H1 polypeptides listed in Table 2 (e.g., SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90).

[0100] In certain configurations, the CAR contains an anti-TROP2 VHH polypeptide containing a hypervariable region H2 (HVR-H2) selected from the HVR-H2 polypeptides in Table 2 (e.g., SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91). In some embodiments, the anti-TROP2 VHH polypeptide provided herein contains at least 80% identical HVR-H2 polypeptides to the HVR-H2 polypeptides listed in Table 2 (e.g., SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91). In some embodiments, the anti-TROP2 VHH polypeptide provided herein contains at least 85% identical HVR-H2 polypeptides to the HVR-H2 polypeptides listed in Table 2 (e.g., SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 90% identical HVR-H2 polypeptides to the HVR-H2 polypeptides listed in Table 2 (e.g., SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91).In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H2 polypeptides to the HVR-H2 polypeptides listed in Table 2 (e.g., SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91).

[0101] In certain configurations, the CAR contains an anti-TROP2 VHH polypeptide with a hypervariable region H3 (HVR-H3) selected from the HVR-H3 polypeptides in Table 2 (e.g., SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 92). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 80% identical HVR-H3 polypeptides to the HVR-H3 polypeptides listed in Table 2 (e.g., SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 92). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 85% identical HVR-H3 polypeptides to the HVR-H3 polypeptides listed in Table 2 (e.g., SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 92). In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least 90% identical HVR-H3 polypeptides to the HVR-H3 polypeptides listed in Table 2 (e.g., SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 92).In some embodiments, the anti-TROP2 VHH polypeptides provided herein contain at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical HVR-H3 polypeptides to the HVR-H3 polypeptides listed in Table 2 (e.g., SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, SEQ ID NO: 92).

[0102] In certain configurations, CAR contains anti-TROP2 ScFv. In some embodiments, anti-TROP2 ScFv contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 101. In some embodiments, anti-TROP2 ScFv contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 101. In some embodiments, anti-TROP2 ScFv contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 101. In some embodiments, anti-TROP2 ScFv contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 101. In some embodiments, anti-TROP2 ScFv contains the polypeptide of SEQ ID NO: 101.

[0103] In certain configurations, the CAR contains an anti-TROP2 ScFv, and the ScFv contains a variable heavy chain (VH) domain and a variable light chain (VL) domain. In some embodiments, the VH domain contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 93. In some embodiments, the VH domain contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 93. In some embodiments, the VH domain contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 93. In some embodiments, the VH domain contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 93. In some embodiments, the VH domain contains the polypeptide of SEQ ID NO: 93. In some embodiments, the VL domain contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 97. In some embodiments, the VL domain contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 97. In some embodiments, the VL domain contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 97. In some embodiments, the VL domain contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 97. In some embodiments, the VL domain contains the polypeptide of SEQ ID NO: 97.

[0104] The ScFv VH domain may contain a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide. In some embodiments, HVR-H1 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 94. In some embodiments, HVR-H1 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 94. In some embodiments, HVR-H1 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 94. In some embodiments, HVR-H1 contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 94. In some embodiments, HVR-H1 contains the polypeptide of SEQ ID NO: 94. In some embodiments, HVR-H2 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 95. In some embodiments, HVR-H2 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 95. In some embodiments, HVR-H2 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 95. In some embodiments, HVR-H2 contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 95. In some embodiments, HVR-H2 contains the polypeptide of SEQ ID NO: 95. In some embodiments, HVR-H3 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 96. In some embodiments, HVR-H3 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 96. In some embodiments, HVR-H3 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-H3 comprises a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 96.

[0105] The ScFv VL domain may contain a hypervariable region L1 (HVR-L1) polypeptide, a hypervariable region L2 (HVR-L2) polypeptide, and a hypervariable region L3 (HVR-L3) polypeptide. In some embodiments, HVR-L1 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 98. In some embodiments, HVR-L1 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 98. In some embodiments, HVR-L1 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 98. In some embodiments, HVR-L1 contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 98. In some embodiments, HVR-L1 contains the polypeptide of SEQ ID NO: 98. In some embodiments, HVR-L2 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-L2 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-L2 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-L2 contains a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-L2 contains the polypeptide of SEQ ID NO: 99. In some embodiments, HVR-L3 contains a polypeptide that is at least about 80% identical to the polypeptide of SEQ ID NO: 100. In some embodiments, HVR-L3 contains a polypeptide that is at least about 85% identical to the polypeptide of SEQ ID NO: 100. In some embodiments, HVR-L3 contains a polypeptide that is at least about 90% identical to the polypeptide of SEQ ID NO: 100. In some embodiments, HVR-L3 comprises a polypeptide that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 100.

[0106] In some embodiments, the CAR comprises an anti-TROP2 VHH polypeptide including a hypervariable region H1 (HVR-H1), a hypervariable region H2 (HVR-H2), and / or a hypervariable region H3 (HVR-H3) selected from the HVR-H1, HVR-H2, and HVR-H3 polypeptides of Table B. [Table B-1] [Table B-2] [Table B-3] [Table B-4]

[0107] The CARs described herein may comprise one or more components selected from (i) one or more signaling domains; (ii) a transmembrane domain; (iii) a hinge; (iv) a cytokine (e.g., IL-15); (v) a T2A sequence; and (vi) one or more linkers. In some embodiments, the CAR comprises a 4-1BB signaling domain. In some embodiments, the CAR comprises a CD3ζ signaling domain. In some embodiments, the CAR comprises a 4-1BB signaling domain and a CD3ζ signaling domain. In some embodiments, the CAR comprises a CD8α hinge. In some embodiments, the intracellular domain comprises a 4-1BB signaling domain, a CD3ζ signaling domain, a T2A sequence, and IL-15. In some embodiments, the CAR comprises, in order (listed from the extracellular domain to the intracellular domain), a TROP2 antigen-binding domain, a linker, a PD-L1 antigen-binding domain, a hinge, a transmembrane domain, a 4-1BB signaling domain, a CD3ζ signaling domain, a T2A sequence, and IL-15. In some embodiments, the CAR comprises, in order (listed from the extracellular domain to the intracellular domain), a PD-L1 antigen-binding domain, a linker, a TROP2 antigen-binding domain, a hinge, a transmembrane domain, a 4-1BB signaling domain, a CD3ζ signaling domain, a T2A sequence, and IL-15.

[0108] In some embodiments, the CAR comprises one or more cytokines. In some embodiments, the CAR comprises one or more interleukins. For example, the CAR may comprise IL-15 (e.g., IL-15 comprising the amino acid sequence of SEQ ID NO: 131, or a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 131).

[0109] In some embodiments, the CAR includes a hinge. In some embodiments, the CAR includes a CD8α hinge. In some embodiments, the CAR includes the polypeptide of SEQ ID NO: 137, or a CD8α hinge comprising a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 137.

[0110] In some embodiments, the CAR includes a transmembrane domain. In some embodiments, the CAR includes a CD8α transmembrane domain. In some embodiments, the CAR includes a CD8α transmembrane domain comprising the polypeptide of SEQ ID NO: 138 or a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 138.

[0111] In some embodiments, the CAR comprises one or more signaling domains. In some embodiments, the CAR comprises one or more signaling domains selected from the 4-1BB signaling domain and the CD3ζ signaling domain. In some embodiments, the CAR comprises a 4-1BB signaling domain comprising the polypeptide of SEQ ID NO: 139, or a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the CAR comprises a CD3ζ signaling domain comprising the polypeptide of SEQ ID NO: 140, or a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 140.

[0112] In some embodiments, the CAR comprises a T2A component. In some embodiments, the CAR comprises a T2A component comprising the polypeptide of SEQ ID NO: 141, or a polypeptide that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 141.

[0113] In this specification, percent amino acid sequence identity to a reference CAR, VHH, VH, VL, HVR, ScFv, cytokine, or other CAR component polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences to achieve maximum percent sequence identity and introducing gaps as necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the scope of the skills of the art, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0114] Nucleic acids, vectors, and recombination methods Nucleic acid constructs encoding CARs as described herein and their components are also provided herein. In some embodiments, the nucleic acid comprises a first polynucleotide encoding a first antigen-binding domain having binding specificity to TROP2 (e.g., anti-TROP2 VHH polypeptide; anti-TROP2 ScFv polypeptide). In some embodiments, the nucleic acid comprises a second polynucleotide encoding a second antigen-binding domain having binding specificity to PD-L1 (e.g., anti-PD-L1 VHH polypeptide; anti-PD-L1 ScFv polypeptide). In some embodiments, the nucleic acid comprises a first polynucleotide encoding a first antigen-binding domain having binding specificity to TROP2 (e.g., anti-TROP2 VHH polypeptide; anti-TROP2 ScFv polypeptide) and a second polynucleotide encoding a second antigen-binding domain having binding specificity to PD-L1 (e.g., anti-PD-L1 VHH polypeptide; anti-PD-L1 ScFv polypeptide). Suitable polynucleotides encoding one or more CAR antigen-binding domains described herein may include commercially available polynucleotides, custom-synthesized polynucleotides (e.g., available from Twist Bioscience in San Francisco, California), and / or polynucleotides encoding one or more variant polypeptides described herein.

[0115] In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an anti-PD-L1 VHH amino acid sequence. In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an anti-PD-L1 VHH amino acid sequence selected from the VHH amino acid sequences in Table 1 (e.g., SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9). In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an amino acid sequence that is at least approximately 80% identical to the anti-PD-L1 VHH amino acid sequences in Table 1. In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an amino acid sequence that is at least approximately 85% identical to the anti-PD-L1 VHH amino acid sequences in Table 1. In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an amino acid sequence that is at least approximately 90% identical to the anti-PD-L1 VHH amino acid sequences in Table 1. In certain configurations, the CAR nucleic acid construct contains a polynucleotide encoding an amino acid sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the anti-PD-L1 VHH amino acid sequence in Table 1.

[0116] In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an anti-TROP2 VHH amino acid sequence. In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an anti-TROP2 VHH amino acid sequence selected from the VHH amino acid sequences in Table 2 (e.g., SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89). In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an amino acid sequence that is at least approximately 80% identical to the anti-TROP2 VHH amino acid sequences in Table 2. In certain configurations, the CAR nucleic acid construct includes a polynucleotide encoding an amino acid sequence that is at least approximately 85% identical to the anti-TROP2 VHH amino acid sequences in Table 2. In certain configurations, the CAR nucleic acid construct contains a polynucleotide encoding an amino acid sequence that is at least approximately 90% identical to the anti-TROP2 VHH amino acid sequence in Table 2. In certain configurations, the CAR nucleic acid construct contains a polynucleotide encoding an amino acid sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the anti-TROP2 VHH amino acid sequence in Table 2.

[0117] In some embodiments, the nucleic acid construct further comprises one or more polynucleotides encoding one or more components selected from (i) one or more signaling domains; (ii) a transmembrane domain; (iii) a hinge; (iv) IL-15; (v) a T2A sequence; and (vi) one or more linkers. In some embodiments, the nucleic acid construct comprises a polynucleotide encoding a 4-1BB signaling domain. In some embodiments, the nucleic acid construct comprises a polynucleotide encoding a CD3ζ signaling domain. In some embodiments, the nucleic acid construct comprises a polynucleotide encoding a 4-1BB signaling domain and a polynucleotide encoding a CD3ζ signaling domain. In some embodiments, the nucleic acid construct comprises a polynucleotide encoding a CD8α hinge. Suitable polynucleotides encoding one or more CAR components described herein may include commercially available polynucleotides, custom synthetic polynucleotides (e.g., available from Twist Bioscience in San Francisco, California), and / or polynucleotides encoding one or more variant polypeptides described herein.

[0118] For the recombinant production of CAR or its components, nucleic acids encoding the CAR or its components can be isolated and inserted into a replicable vector for further cloning and / or expression. Any suitable vector can be used. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0119] Methods for genetically modifying gamma delta T cells to incorporate the CAR-encoding nucleic acids described herein may include any techniques known to those skilled in the art. Suitable methodologies include, but are not limited to, lentiviral transduction, retroviral transduction, adenovirus transduction, cell transfection by electroporation, lipid-based transfection reagents, nanoparticles, calcium chloride-based transfection methods, or bacterial transposons, mRNA, nucleic acids, and ribonucleoproteins.

[0120] In embodiments in which lentiviruses / retroviruses / adenoviruses can be used for transduction, the inclusion of chemical reagents as understood by those skilled in the art may be used to enhance this process. These include, but are not limited to, hexadimethrin bromide (Polybrene), recombinant human fibronectin (Retronectin - Takara Clontec, etc.), bectofucin (Milteny Biotech), and TRANSPLUS viral transduction enhancers (ALSTEM Cell Advancements).

[0121] Detection of the efficiency of transduction and expression of CAR constructs by gamma delta T cells may include, but are not limited to, any techniques known to those skilled in the art, quantitative PCR and antibody-based detection methods, such as flow cytometry or Western blotting.

[0122] Genome modification In some embodiments, modified gamma delta T cells include one or more genomic modifications. Genomic modifications may include disruption of genes (e.g., gene knockout (KO)) introduced by an appropriate genome editing process (e.g., CRISPR-Cas9; CRISPR-Cas12i). In some embodiments, the genomic modification is the disruption of genes in the TGFβ signaling pathway. TGFβ signaling generally controls proliferation, cell differentiation, and other functions in various cell types, and can play a role in cell cycle control, immune system regulation, and development in specific cell types. Disruption of TGFβ signaling genes may include disruption (e.g., knockout) of any genes encoding TGFβ signaling pathway proteins and / or members of the TGFβ superfamily, including TGFβ superfamily type II receptors, type I serine / threonine kinase receptors, type II serine / threonine kinase receptors, TGFβ type I receptors, TGFβ type II receptors, activin receptors, nodal receptors, activin / nodal receptors, activin receptor-like kinases (ALK; e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, and ALK8), and downstream effectors, e.g., R-SMAD and other SMAD proteins (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD8 / 9).

[0123] In some embodiments, the modified gamma delta T cells include genomic modifications in the TGFβ2 receptor. In some embodiments, the TGFβ2 receptor gene is knocked out or substantially knocked out in the modified gamma delta T cells. In some embodiments, a population of modified gamma delta T cells including TGFβ2 receptor knockout expresses the TGFβ2 receptor at undetectable levels. In some embodiments, a population of modified gamma delta T cells including TGFβ2 receptor knockout expresses the TGFβ2 receptor at low levels (e.g., less than 10%, less than 5%, less than 3%).

[0124] The introduction of genomic modifications (e.g., TGFβ2 receptor knockout) may be performed in quiescent or activated gamma delta T cells. The introduction of genomic modifications (e.g., TGFβ2 receptor knockout) may be performed before or after expansion of gamma delta T cells (e.g., by the expansion process described herein).

[0125] Treatment method Methods for treating infection or cancer in an individual are provided herein, comprising the step of providing the aforementioned individual with gamma delta T cells (e.g., modified gamma delta T cells). In some embodiments, the gamma delta T cells are obtained from different individuals (allogeneic treatment). Thus, donor gamma delta T cells are used, for example, for treating infections of viruses, bacteria, fungi, or protozoa, or for treating cancer in a recipient subject where the donor and recipient are not from the same individual. As understood, prior to providing the gamma delta T cells to a second subject, these gamma delta T cells are activated and selectively expanded according to the methods described herein. In some embodiments, the gamma delta T cells are modified as discussed herein to provide CAR-modified gamma delta T cells. In some embodiments, the gamma delta T cells are further modified to provide TGFβ knockout gamma delta T cells.

[0126] A process for autodonating gamma delta T cells to a subject is also provided, comprising the steps of obtaining a sample of gamma delta T cells from the subject and culturing the gamma delta T cells to enable administration back to the subject, wherein the culturing step includes a step of expanding and modifying the gamma delta T cells as described herein.

[0127] Methods of administration for providing gamma delta T cells to a recipient may include, for example, intravenous, intradermal, intraperitoneal, intrathecal, intratumoral, or subcutaneous injection. Administration may be site-administered or systemic to the individual. The administration method may be prophylactic or therapeutic, and a “prophylactic effective dose” or “therapeutic effective dose” of gamma delta T cells sufficient to provide benefit to the individual will be provided. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, such as determining the dosage, is the responsibility of the general practitioner and other physicians.

[0128] In some embodiments, the gamma delta T cells described herein are provided for the treatment of subjects having cancer. In some embodiments, the cancer is characterized as a solid tumor-type carcinoma. In some embodiments, the cancer is characterized as an epithelial tissue carcinoma. In some embodiments, the cancer is characterized as a squamous cell tumor-type carcinoma. In some embodiments, the cancer may include, but is not limited to, pancreatic cancer, gastric cancer, renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer), breast cancer, colon cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer (e.g., prostatic adenocarcinoma), endometrioid carcinoma, oral squamous cell carcinoma, and papillary thyroid cancer.

[0129] Pharmaceutical compositions comprising modified gamma delta T cells as described herein are also provided herein. In some embodiments, the pharmaceutical composition comprises modified gamma delta T cells in a dose suitable for administration to an individual to provide a therapeutic effect. In some embodiments, the pharmaceutical composition further comprises one or more therapeutic agents selected from antibody immunotherapies, chemotherapeutic agents, biological agents, cytokines, or combinations thereof.

[0130] Anti-TROP2 agents and anti-PD-L1 agents Agents that bind to TROP2 or a portion thereof are provided herein. Agents that bind to TROP2 or a portion thereof may be called anti-TROP2 agents and may include anti-TROP2 nanobodies (e.g., VHH antibodies), anti-TROP2 nanobody fragments (e.g., antigen-binding fragments), anti-TROP2 nanobody derivatives, anti-TROP2 antibodies, anti-TROP2 antibody fragments (e.g., antigen-binding fragments), and anti-TROP2 antibody derivatives. In some embodiments, anti-TROP2 agents are isolated (e.g., isolated from components of their natural environment (e.g., animals, biological samples)). In some embodiments, anti-TROP2 agents do not exist in nature (e.g., are produced by human intervention). In some embodiments, the anti-TROP2 agent is a humanized nanobody, a humanized antibody, or an antigen-binding fragment thereof. In some embodiments, anti-TROP2 is a derivative of a humanized nanobody or a humanized antibody. In some embodiments, the anti-TROP2 agent binds to TROP2 under laboratory conditions (e.g., binds to TROP2 in vitro, binds to TROP2 in a flow cytometry assay, binds to TROP2 in an ELISA). In some embodiments, the anti-TROP2 agent binds to TROP2 under physiological conditions (e.g., by binding to TROP2 in the target cells).

[0131] In some embodiments, the anti-TROP2 agent comprises a VHH polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-TROP2 agent comprises an HVR-H1 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-TROP2 agent comprises an HVR-H2 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-TROP2 agent comprises an HVR-H3 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein.

[0132] Agents that bind to PD-L1 or a portion thereof are also provided herein. Agents that bind to PD-L1 or a portion thereof may be called anti-PD-L1 agents and may include anti-PD-L1 nanobodies (e.g., VHH antibodies), anti-PD-L1 nanobodyle fragments (e.g., antigen-binding fragments), anti-PD-L1 nanobodyle derivatives, anti-PD-L1 antibodies, anti-PD-L1 antibody fragments (e.g., antigen-binding fragments), and anti-PD-L1 antibody derivatives. In some embodiments, anti-PD-L1 agents are isolated (e.g., isolated from components of their natural environment (e.g., animals, biological samples)). In some embodiments, anti-PD-L1 agents do not exist naturally (e.g., are produced by human intervention). In some embodiments, anti-PD-L1 agents are humanized nanobodies, humanized antibodies, or antigen-binding fragments thereof. In some embodiments, anti-PD-L1 is a derivative of a humanized nanobody or humanized antibody. In some embodiments, the anti-PD-L1 agent binds to PD-L1 under laboratory conditions (e.g., binding to PD-L1 in vitro, binding to PD-L1 in flow cytometry assays, and binding to PD-L1 in ELISA). In some embodiments, the anti-PD-L1 agent binds to PD-L1 under physiological conditions (e.g., binding to PD-L1 in cells of interest).

[0133] In some embodiments, the anti-PD-L1 agent comprises a VHH polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-PD-L1 agent comprises an HVR-H1 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-PD-L1 agent comprises an HVR-H2 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein. In some embodiments, the anti-PD-L1 agent comprises an HVR-H3 polypeptide provided herein, or a polypeptide that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide provided herein.

[0134] In some embodiments, the agents described herein include one or more modifications. For example, immunoconjugates comprising the agents or antibodies described herein can be conjugated to toxins (e.g., enzymatically active toxins or fragments thereof of bacterial, fungal, plant, or animal origin), radioisotopes (e.g., radioconjugates), or cytotoxic agents such as cytotoxic drugs. Such conjugates are sometimes referred to as antibody-drug conjugates or ADCs. Conjugates can be prepared using any suitable bifunctional protein coupling agent, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., torylene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, the drug may be conjugated to cells (e.g., chemically conjugated). Such conjugates are sometimes called antibody-cell conjugates (ACCs). Drug-cell conjugation can be direct or indirect (e.g., by a nucleic acid linker). In some embodiments, the drug may be conjugated to immune cells (e.g., T cells, NK cells, macrophages, etc.).

[0135] In some embodiments, the drugs described herein include one or more detectable markers or labels. For example, for research and diagnostic purposes, the drugs described herein may be labeled with a detectable portion. Any suitable marker, label, or portion may be associated with or conjugated to the drugs described herein. In some embodiments, the drugs are, for example, 35 S, 14 C, 125 l,3 H, and 131 The drug is labeled with one or more radioisotopes, such as l. The drug can be labeled with radioisotopes using techniques known in the art, and its radioactivity can be measured, for example, using scintillation counting. In some embodiments, the drug is labeled with one or more fluorescent labels, such as rare earth chelates (europium chelate), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine, phycoerythrin (PE), Texas Red, and Brilliant Violet™. The fluorescent labels can be conjugated to the drug using methods known in the art. The fluorescence can be quantified, for example, using a flow cytometer, imaging microscope, or fluorophotometer.

[0136] Pharmaceutical formulations, administration, and routes of administration Therapeutic compositions comprising an anti-TROP2 agent described herein and pharmaceutically acceptable excipients are provided herein. Therapeutic compositions comprising an anti-PD-L1 agent described herein and pharmaceutically acceptable excipients are also provided herein. In some embodiments, anti-TROP2 agents and / or anti-PD-L1 agents or their antigen-binding fragments can be formulated into pharmaceutical compositions useful for a variety of purposes, including the treatment of diseases or disorders (e.g., cancer). Pharmaceutical compositions comprising one or more antibodies can be administered to patients in need using a pharmaceutical device, and according to one embodiment of the art, a kit comprising such a device is provided. Such devices and kits may be designed for the routine administration (including self-administration) of the pharmaceutical compositions herein.

[0137] Therapeutic compositions comprising an anti-TROP2 agent and / or anti-PD-L1 agent as described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer are provided herein. Therapeutic formulations of anti-TROP2 agents and / or anti-PD-L1 agents can be prepared for storage by mixing the agent of desired purity with a physiologically and / or pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution. The acceptable carrier, excipient, or stabilizer is nontoxic to the recipient at the dose and concentration used and includes buffers such as phosphates, citrates, and other organic acids. Antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; polyvinylpyrrolid Hydrophilic polymers such as Zn; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0138] The formulations described herein may also contain two or more active compounds, preferably those having complementary activities that do not adversely affect each other, as necessary for the specific indication being treated. Such molecules are appropriately present in combination in amounts effective for the intended purpose. Formulations for in vivo administration are generally sterile. This can be achieved, for example, by filtration with a sterile filtration membrane.

[0139] Sustained-release formulations may be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the drug, which may be in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as Lupron Depot® (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release for more than 100 days, while certain hydrogels release proteins over shorter periods.

[0140] For therapeutic use, the anti-TROP2 and / or anti-PD-L1 agents provided herein may be administered to mammals, e.g., humans, in pharmaceutically acceptable dosage forms, including those discussed above, which may be administered to humans intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intradermal, intra-articular, intrasynovial, intrathecal, intratumoral, oral, topical, or inhalation routes. For the prevention or treatment of disease, the appropriate dosage of the agent depends on the type of disease being treated, the severity and course of the disease, whether the agent is administered for prophylactic or treatment purposes, previous treatments, the patient's medical history and response to the agent, and the discretion of the healthcare professional. The agent may be administered to the patient in a single dose or over a series of treatments.

[0141] In some embodiments, compositions containing the agents described herein can be administered as monotherapy, and in some embodiments, compositions containing the agents can be administered as part of combination therapy. Accordingly, therapeutic compositions in which the agents are used as adjuvants or in combination with adjuvants are provided herein. In some cases, the effectiveness of an agent in the prevention or treatment of a disease may be improved by administering the agent sequentially or in combination with another agent effective for those purposes, such as a chemotherapeutic agent for the treatment of cancer or microbial infections. In other cases, the agent may help to enhance or sensitize cells to chemotherapeutic treatment, thus enabling effectiveness at lower doses and with lower toxicity. Certain combination therapies include, in addition to administering a composition containing the agents described herein, delivering a second therapeutic regimen selected from the group consisting of chemotherapeutic agents, radiotherapy, surgery, and any combination thereof. Such other agents may be present in the administered composition or administered separately. The agent may also be appropriately administered sequentially or in combination with other agents or modalities, such as chemotherapeutic agents or radiotherapy for the treatment of cancer, infections, etc., or immunosuppressants.

[0142] Research and diagnosis Diagnostic reagents comprising anti-TROP2 agents described herein are provided herein. For example, anti-TROP2 agents provided herein can be used to detect and / or purify TROP2 from body fluids or tissues. For example, anti-TROP2 agents may be useful for diagnostic assays of TROP2, for example, for detecting its presence in specific cells, tissues, or body fluids. Such diagnostic methods may be useful, for example, for diagnosing hyperproliferative diseases or disorders. Methods for detecting and / or measuring TROP2 levels in a sample of or from a subject are also provided herein. For example, a method may include contacting a sample (e.g., a biological sample known or suspected to contain TROP2) with a drug provided herein, and, if the sample contains TROP2, detecting a TROP2:drug complex. In some embodiments, the TROP2 detection method is performed in vitro. In some embodiments, the TROP2 detection method is performed in vivo. For in vivo diagnostic assays, the drug is combined with a radionuclide ( 111 In, 99 Tc, 14 C, 131 l, 125 l, 3 H, 32 P, or 35 It can be marked with an S, etc.

[0143] Reagents containing the anti-TROP2 agents described herein for non-diagnostic use are also provided herein. Reagents containing the anti-TROP2 agents described herein for non-therapeutic use are also provided herein. Reagents containing the anti-TROP2 agents described herein for non-diagnostic and non-therapeutic use are also provided herein. For example, reagents containing the anti-TROP2 agents described herein for use in research applications are provided herein. Research applications may include investigating TROP2 and its role in cell signaling, signal transduction, cell migration, cell proliferation, development, disease, infection, inflammation, tissue remodeling, tumor growth, tumor angiogenesis, tumor proliferation, tumor migration, tumor invasion, tumor radiation resistance, metastasis, etc. Methods for detecting TROP2 in non-biological samples and / or measuring TROP2 levels are also provided herein. For example, a method may include contacting a non-biological sample (e.g., a laboratory study sample known or suspected to contain TROP2) with an anti-TROP2 agent provided herein, and detecting a TROP2:drug complex if the sample contains TROP2. Laboratory research samples may include non-human animal models, samples from non-human animal models, cell lines, and products produced by cell lines.

[0144] The anti-TROP2 agents and antibodies provided herein can be used in any suitable detection assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0145] Diagnostic reagents comprising anti-PD-L1 agents described herein are also provided herein. For example, anti-PD-L1 agents provided herein may be used to detect and / or purify PD-L1 from body fluids or tissues. For example, anti-PD-L1 agents may be useful in diagnostic assays for PD-L1, e.g., for detecting its presence in specific cells, tissues or body fluids. Such diagnostic methods may be useful, for example, for diagnosing hyperproliferative diseases or disorders. Methods for detecting PD-L1 in or from a sample of a subject and / or measuring PD-L1 levels are also provided herein. For example, a method may include contacting a sample (e.g., a biological sample known or suspected to contain TROP2) with a drug provided herein, and, if the sample contains PD-L1, detecting a PD-L1:drug complex. In some embodiments, the PD-L1 detection method is performed in vitro. In some embodiments, the PD-L1 detection method is performed in vivo. For in vivo diagnostic assays, the drug is bound to a radionuclide ( 111 In, 99 Tc, 14 C, 131 l, 125 l, 3 H, 32 P, or 35 It can be marked with an S, etc.

[0146] Reagents containing the anti-PD-L1 agents described herein for non-diagnostic use are also provided herein. Reagents containing the anti-PD-L1 agents described herein for non-therapeutic use are also provided herein. Reagents containing the anti-PD-L1 agents described herein for non-diagnostic and non-therapeutic use are also provided herein. For example, reagents containing the anti-PD-L1 agents described herein for use in research applications are provided herein. Research applications may include investigating PD-L1 and its role in cell signaling, signal transduction, cell migration, cell proliferation, development, disease, infection, inflammation, tissue remodeling, tumor growth, tumor angiogenesis, tumor growth, tumor migration, tumor invasion, tumor radiation resistance, metastasis, etc. Methods for detecting PD-L1 in non-biological samples and / or measuring PD-L1 levels are also provided herein. For example, the method may include contacting a non-biological sample (e.g., a laboratory study sample known or suspected to contain PD-L1) with an anti-PD-L1 agent provided herein, and, if the sample contains PD-L1, detecting a PD-L1:agent complex. Laboratory study samples may include non-human animal models, samples from non-human animal models, cell lines, and products produced by cell lines.

[0147] The anti-PD-L1 agents and antibodies provided herein may be used in any suitable detection assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0148] Reagents containing the CARs described herein for non-therapeutic use are also provided herein. For example, reagents containing the CARs described herein for use in research applications are provided herein. Research applications may include investigating PD-L1 and / or TROP2 and their roles in cell signaling, signal transduction, cell migration, cell proliferation, development, disease, infection, inflammation, tissue remodeling, tumor growth, tumor angiogenesis, tumor proliferation, tumor migration, tumor invasion, tumor radiation resistance, metastasis, etc.

[0149] kit In certain embodiments, a kit is provided. The kit may contain any suitable combination of any components and compositions described herein that are useful for carrying out any of the methods described herein. The kit may further contain any reagents, media, buffers, or other components that are useful for carrying out any of the methods described herein. For example, the kit may contain cell culture media and supplements suitable for culturing T cells (e.g., CTS® OpTmizer® T cell expansion medium (ThermoFisher Scientific) supplemented with ICTSR (e.g., 2.5%), p / s (e.g., 1%), GLUTAMAX (e.g., 2 mM) and human AB serum (e.g., 2.5% of 1 million cells per mL). In some embodiments, the kit includes a binder comprising (i) a differentiation cluster 3 (CD3) binder (e.g., an antibody or fragment thereof) and (ii) a differentiation cluster 2 (CD2) binder (e.g., an antibody or fragment thereof). In some embodiments, the kit includes a binder comprising a CD3 binder and a CD2 binder. In some embodiments, the kit includes one or more cytokines. In some embodiments, the kit includes one or more cytokines comprising interleukin-15 (IL-15). In some embodiments, the kit includes one or more cytokines comprising interleukin-15 (IL-15). In some embodiments, the kit includes one or more cytokines comprising interleukin-2 (IL-2). In some embodiments, the kit includes one or more cytokines comprising interleukin-2 (IL-2).

[0150] In some embodiments, the kit includes cells. In some embodiments, the kit includes cells derived from a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, the kit includes cells depleted of alpha-beta T cells.

[0151] The components of the kit may be in separate containers, or multiple components may be in a single container. Suitable containers include single tubes (e.g., vials), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), and so on.

[0152] The kit may also include instructions for performing one or more of the methods described herein and / or descriptions of one or more components described herein. For example, the kit may include instructions for selectively expanding gamma delta T cells ex vivo. The instructions and / or descriptions may be in printed form or included in a kit insert. In some embodiments, the instructions and / or descriptions are provided as electronic storage data files residing on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, diskette, etc. The kit may also include written descriptions of an internet location providing such instructions or descriptions.

[0153] Specific implementation forms The following are non-exclusive examples of specific implementations of this technology.

[0154] A1. A method for selectively expanding gamma delta T cells ex vivo, A method for increasing the number of gamma delta T cells in an original cell population under expansion conditions, thereby generating an expanded gamma delta T cell-enriched cell population, wherein the expansion conditions include a binder selected from two or more of the following: (i) differentiation cluster 3 (CD3) binder, (ii) differentiation cluster 2 (CD2) binder, (iii) NKp46 binder, (iv) NKp44 binder, and (v) NKp30 binder.

[0155] A2. The method according to Embodiment A1, wherein the expansion condition includes a CD3 binder and a CD2 binder.

[0156] A2.1 The method according to Embodiment A1, wherein the expansion condition includes a binder comprising a CD3 binder and a CD2 binder.

[0157] A3. The method according to any one of Embodiments A1 to A2, further comprising one or more cytokines as expansion conditions.

[0158] A4. The method according to Embodiment A3, wherein one or more cytokines include interleukin 15 (IL-15) and / or interleukin 2 (IL-2).

[0159] A5. The method according to Embodiment A3, wherein one or more cytokines consist of interleukin 15 (IL-15).

[0160] A5.1 The method according to Embodiment A3, wherein one or more cytokines consist of interleukin-2 (IL-2).

[0161] A6. The method according to any one of Embodiments A1 to A5.1, wherein the origin cell population is derived from a population of peripheral blood mononuclear cells (PBMCs).

[0162] A7. The method according to any one of embodiments A1 to A6, wherein alpha-beta T cells are depleted from the origin cell population.

[0163] A8. The method according to any one of embodiments A1 to A7, wherein the CD3 conjugate is an antibody or a fragment thereof.

[0164] A9. The method according to any one of Embodiments A1 to A8, wherein the CD2 conjugate is an antibody or a fragment thereof.

[0165] A10. The method according to any one of Embodiments A1 to A9, wherein the cells are enlarged by approximately 200 to 300 times in the enlarged gamma delta T cell-enriched cell population compared to the origin cell population.

[0166] A11. The method according to any one of Embodiments A1 to A10, wherein the expanded gamma delta T cell enriched cell population includes multiple subsets of gamma delta T cells.

[0167] A12. The method according to Embodiment A11, wherein multiple subsets of gamma delta T cells include Vdelta1 (Vδ1) T cells and Vdelta2 (Vδ2) T cells.

[0168] A13. The method according to Embodiment A11 or A12, wherein multiple subsets of gamma delta T cells include Vdelta1 (Vδ1) T cells, Vdelta2 (Vδ2) T cells, and non-Vδ1 and Vδ2 T cells.

[0169] A14. The method according to Embodiment A12 or A13, wherein Vδ1 T cells are increased by approximately 1,000 to 10,000 times in the expanded gamma delta T cell-enriched cell population compared to the origin cell population.

[0170] A15. The method according to any one of embodiments A12 to A14, wherein Vδ2 T cells are increased by approximately 100 to 1,400 times in the expanded gamma delta T cell-enriched cell population compared to the origin cell population.

[0171] A15.1 The method according to any one of Embodiments A12 to A14, wherein Vδ2 T cells are enlarged by approximately 350 times or more in the enlarged gamma delta T cell-enriched cell population compared to the origin cell population.

[0172] A16. The method according to any one of Embodiments A1 to A15.1, wherein the expanded gamma delta T cell enriched cell population contains approximately 90% or more gamma delta T cells.

[0173] A17. The method according to any one of Embodiments A1 to A16, wherein the expanded gamma delta T cell enriched cell population contains approximately 95% or more gamma delta T cells.

[0174] A18. The method according to any one of Embodiments A1 to A17, wherein the expanded gamma delta T cell enriched cell population comprises approximately 10–55% Vδ1 T cells, approximately 30–80% Vδ2 T cells, and approximately 5–15% non-Vδ1 and Vδ2 T cells.

[0175] A18.1 The method according to any one of Embodiments A1 to A17, wherein the expanded gamma delta T cell enriched cell population comprises approximately 50–55% Vδ1 T cells, approximately 28–33% Vδ2 T cells, and approximately 6–11% non-Vδ1 and Vδ2 T cells.

[0176] A19. The method according to any one of Embodiments A1 to A18.1, wherein the expanded gamma delta T cell-enriched cell population contains approximately 5% or less natural killer (NK) cells.

[0177] A20. The method according to any one of Embodiments A1 to A19, wherein the expanded gamma delta T cell-enriched cell population contains approximately 3% or less natural killer (NK) cells.

[0178] A21. The method according to any one of Embodiments A1 to A20, wherein the expanded gamma-delta T cell enriched cell population contains about 5% or less alpha-beta T cells.

[0179] A22. The method according to any one of Embodiments A1 to A21, wherein the expanded gamma-delta T cell enriched cell population contains about 3% or less alpha-beta T cells.

[0180] A22.1 The method according to any one of Embodiments A1 to A22, wherein the expanded gamma-delta T cell enriched cell population contains about 0.5% or less alpha-beta T cells.

[0181] A22.2 The method according to any one of Embodiments A1 to A22.1, wherein the expanded gamma-delta T cell enriched cell population contains about 0.1% or less of alpha-beta T cells.

[0182] A23. The method according to any one of Embodiments A1 to A22.2, further comprising isolating gamma delta T cells from an expanded gamma delta T cell-enriched cell population.

[0183] A24. The method according to any one of Embodiments A1 to A23, further comprising storing an expanded gamma delta T cell-enriched cell population or an isolate thereof in a cell bank.

[0184] A25. The method according to any one of Embodiments A1 to A24, further comprising transfecting or transfecting gamma delta T cells with the nucleic acid described in any one of Embodiments D1 to D18 or the vector described in any one of Embodiments D21 to D23 before expansion.

[0185] A26. The method according to any one of Embodiments A1 to A25, further comprising introducing a genomic modification into cells in an expanded gamma delta T cell-enriched cell population by a genome editing process.

[0186] A27. The method according to Embodiment A26, wherein the genome modification is a disrupted gene encoding TGF-beta receptor 2.

[0187] A28. An expanded gamma delta T cell-enriched cell population produced by the method described in any one of Embodiments A1 to A27.

[0188] A29. Embodiment for the production of genetically modified cells: Use of an expanded gamma delta T cell enriched cell population as in A28.

[0189] A30. Use of an expanded gamma delta T cell enriched cell population of Embodiment A28 or A29 for treatment of subjects requiring it.

[0190] A31. Use according to Embodiment A30, in which the subject has cancer.

[0191] A32. Use according to Embodiment A30 or A31, wherein the subject has a solid tumor or carcinoma.

[0192] A33. Use according to any one of Embodiments A30 to A32, wherein the subject is one or more of pancreatic cancer, gastric cancer, renal cell carcinoma, lung cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, endometrioid carcinoma, oral squamous cell carcinoma, and papillary thyroid cancer.

[0193] B1. A kit for selectively expanding gamma delta T cells ex vivo, comprising two or more conjugates selected from (i) differentiation cluster 3 (CD3) conjugates, (ii) differentiation cluster 2 (CD2) conjugates, (iii) NKp46 conjugates, (iv) NKp44 conjugates, and (v) NKp30 conjugates.

[0194] B2. The kit according to Embodiment B1, wherein the binder comprises a CD3 binder and a CD2 binder.

[0195] B2.1 The kit according to Embodiment B1, wherein the binder consists of a CD3 binder and a CD2 binder.

[0196] B3. A kit according to any one of embodiments B1 to B2, further comprising one or more cytokines.

[0197] B4. The kit according to Embodiment B3, wherein one or more cytokines include interleukin-15 (IL-15) and / or interleukin-2 (IL-2).

[0198] B5. The kit according to Embodiment B3, wherein one or more cytokines consist of interleukin 15 (IL-15).

[0199] B5.1 The kit according to Embodiment B3, wherein one or more cytokines consist of interleukin-2 (IL-2).

[0200] B6. The kit according to any one of Embodiments B1 to B5.1, further comprising cells derived from a population of peripheral blood mononuclear cells (PBMCs).

[0201] B7. The kit according to embodiment B6, in which alpha-beta T cells are depleted from the cells.

[0202] B8. The kit according to any one of embodiments B1 to B7, wherein the CD3 conjugate is an antibody or a fragment thereof.

[0203] B9. The kit according to any one of embodiments B1 to B8, wherein the CD2 conjugate is an antibody or a fragment thereof.

[0204] C1. Modified gamma delta T cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a first antigen-binding domain having binding specificity to TROP2 and a second antigen-binding domain having binding specificity to PD-L1.

[0205] C1.1 Modified gamma delta T cells according to Embodiment C1, wherein the first antigen-binding domain specifically binds to the target of TROP2.

[0206] C1.2 Modified gamma delta T cells according to Embodiment C1, wherein the first antigen-binding domain specifically binds to two or more targets of TROP2.

[0207] C1.3 A modified gamma delta T cell according to any one of embodiments C1 to C1.2, wherein the second antigen-binding domain specifically binds to the target of PD-L1.

[0208] C1.4 Modified gamma delta T cells according to any one of embodiments C1 to C1.2, wherein the second antigen-binding domain specifically binds to two or more targets of PD-L1.

[0209] C2. A modified gamma delta T cell according to any one of embodiments C1 to C1.4, wherein the first antigen-binding domain comprises an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0210] C3. A modified gamma delta T cell according to any one of embodiments C1 to C1.4, wherein the first antigen-binding domain comprises an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0211] C4. A modified gamma delta T cell according to any one of embodiments C1 to C1.4, wherein the first antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, and SEQ ID NO: 89.

[0212] C5. The first antigen-binding domain is a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, and 90; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, and 91; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 88, and 92. A modified gamma delta T cell according to any one of embodiments C1 to C4, including the modified gamma delta T cell described above.

[0213] C6. A modified gamma delta T cell according to any one of embodiments C1 to C5, wherein the second antigen-binding domain comprises an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0214] C7. A modified gamma delta T cell according to any one of embodiments C1 to C5, wherein the second antigen-binding domain comprises an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0215] The modified gamma-delta T cell according to any one of Embodiments C1-C5, wherein the second antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0216] C9. The second antigen-binding domain is a) a hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10; b) a hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11; and c) a hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12 The modified gamma-delta T cell according to any one of Embodiments C1-C8, comprising

[0217] C10. The modified gamma-delta T cell according to any one of Embodiments C1-C9, wherein the CAR comprises a polypeptide that is about 90% or more identical to a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.

[0218] C11. The modified gamma-delta T cell according to any one of Embodiments C1-C9, wherein the CAR comprises a polypeptide that is about 95% or more identical to a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.

[0219] C12. The modified gamma-delta T cell according to any one of Embodiments C1-C9, wherein the CAR comprises a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.

[0220] A modified gamma delta T cell according to any one of embodiments C1 to C12, wherein C13.CAR further comprises one or more components selected from (i) one or more signaling domains; (ii) a transmembrane domain; (iii) a hinge; (iv) IL-15; (v) a T2A sequence; and (vi) a linker.

[0221] C14. Modified gamma delta T cells according to Embodiment C13, wherein one or more signaling domains comprise a 4-1BB signaling domain, a CD3ζ signaling domain, or a 4-1BB signaling domain and a CD3ζ signaling domain.

[0222] C15. Modified gamma delta T cells according to Embodiment C13 or C14, wherein the hinge comprises a CD8α hinge.

[0223] C16. Modified gamma delta T cells according to any one of embodiments C1 to C15, further comprising genome modification.

[0224] C17. Modified gamma delta T cells as described in Embodiment C16, wherein the genome modification is a disrupted gene encoding TGF-beta receptor 2.

[0225] C18. Modified gamma delta T cells according to any one of embodiments C1 to C17 for use in the treatment of subjects requiring such treatment.

[0226] C19. Modified gamma delta T cells according to Embodiment C18, wherein the subject has cancer.

[0227] C20. Modified gamma delta T cells according to Embodiment C18 or C19, wherein the subject has a solid tumor.

[0228] C21. Modified gamma delta T cells according to any one of Embodiments C18 to C20, wherein the target is one or more of pancreatic cancer, gastric cancer, renal cell carcinoma, lung cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, endometrioid carcinoma, oral squamous cell carcinoma, and papillary thyroid carcinoma.

[0229] C22. A modified gamma delta T cell according to any one of embodiments C1 to C21, wherein the first antigen-binding domain comprises a single-stranded variable fragment (ScFv).

[0230] C23.ScFv contains a variable heavy chain (VH) domain and a variable light chain (VL) domain. Here, the VH domain comprises a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide, where HVR-H1 comprises the polypeptide of SEQ ID NO: 94, HVR-H2 comprises the polypeptide of SEQ ID NO: 95, and HVR-H3 comprises the polypeptide of SEQ ID NO: 96. The modified gamma delta T cell according to Embodiment C22, wherein the VL domain comprises a hypervariable region L1 (HVR-L1) polypeptide, a hypervariable region L2 (HVR-L2) polypeptide, and a hypervariable region L3 (HVR-L3) polypeptide, where HVR-L1 comprises the polypeptide of SEQ ID NO: 98, HVR-L2 comprises the polypeptide of SEQ ID NO: 99, and HVR-L3 comprises the polypeptide of SEQ ID NO: 100.

[0231] A modified gamma delta T cell according to Embodiment C23, wherein the C24.VH domain contains the polypeptide of Sequence ID No. 93 and the VL domain contains the polypeptide of Sequence ID No. 97.

[0232] D1. A nucleic acid encoding a chimeric antigen receptor (CAR), comprising (i) a first polynucleotide encoding a first antigen-binding domain having binding specificity to TROP2, and (ii) a second polynucleotide encoding a second antigen-binding domain having binding specificity to PD-L1.

[0233] D1.1 The nucleic acid according to embodiment D1, wherein the first antigen-binding domain specifically binds to a target in TROP2.

[0234] D1.2 The nucleic acid according to Embodiment D1, wherein the first antigen-binding domain specifically binds to two or more targets in TROP2.

[0235] D1.3 The nucleic acid according to any one of embodiments D1 to D1.2, wherein the second antigen-binding domain specifically binds to a target in PD-L1.

[0236] D1.4 The nucleic acid according to any one of embodiments D1 to D1.2, wherein the second antigen-binding domain specifically binds to two or more targets in PD-L1.

[0237] D2. The nucleic acid according to any one of embodiments D1 to D1.4, wherein the first polynucleotide encodes a first antigen-binding domain comprising an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NOs: SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0238] D3. The nucleic acid according to any one of embodiments D1 to D1.4, wherein the first polynucleotide encodes a first antigen-binding domain comprising an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NOs. 13, SEQ ID NOs. 17, SEQ ID NOs. 21, SEQ ID NOs. 25, SEQ ID NOs. 29, SEQ ID NOs. 33, SEQ ID NOs. 37, SEQ ID NOs. 41, SEQ ID NOs. 45, SEQ ID NOs. 49, SEQ ID NOs. 53, SEQ ID NOs. 57, SEQ ID NOs. 61, SEQ ID NOs. 65, SEQ ID NOs. 69, SEQ ID NOs. 73, SEQ ID NOs. 77, SEQ ID NOs. 81, SEQ ID NOs. 85, and SEQ ID NOs. 89.

[0239] D4. The nucleic acid according to any one of embodiments D1 to D1.4, wherein the first polynucleotide encodes a first antigen-binding domain comprising an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, and SEQ ID NO: 89.

[0240] D5. The first polynucleotide is a) a hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, and SEQ ID NO: 90; b) a hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, and SEQ ID NO: 91; and c) a hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 88, and SEQ ID NO: 92 The nucleic acid according to any one of embodiments D1 to D4, encoding a first antigen-binding domain comprising the same.

[0241] D6. The nucleic acid according to any one of embodiments D1 to D5, wherein the second polynucleotide encodes a second antigen-binding domain having an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0242] D7. The nucleic acid according to any one of embodiments D1 to D5, wherein the second polynucleotide encodes a second antigen-binding domain comprising an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0243] D8. The nucleic acid according to any one of embodiments D1 to D5, wherein the second polynucleotide encodes a second antigen-binding domain comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0244] D9. The second polynucleotide is a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 2, 6, and 10; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 3, 7, and 11; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 4, 8, and 12. A nucleic acid according to any one of embodiments D1 to D8, which encodes a second antigen-binding domain including

[0245] The nucleic acid according to any one of Embodiments D1 to D9, wherein D10.CAR comprises a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NOs. 102, SEQ ID NOs. 103, SEQ ID NOs. 104, SEQ ID NOs. 105, SEQ ID NOs. 106, and SEQ ID NOs. 107.

[0246] The nucleic acid according to any one of Embodiments D1 to D9, wherein D11.CAR comprises a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NOs. 102, SEQ ID NOs. 103, SEQ ID NOs. 104, SEQ ID NOs. 105, SEQ ID NOs. 106, and SEQ ID NOs. 107.

[0247] The nucleic acid according to any one of embodiments D1 to D9, wherein D12.CAR comprises a polypeptide selected from SEQ ID NOs. 102, SEQ ID NOs. 103, SEQ ID NOs. 104, SEQ ID NOs. 105, SEQ ID NOs. 106, and SEQ ID NOs. 107.

[0248] D13. The nucleic acid according to any one of embodiments D1 to D12, further comprising one or more polynucleotides encoding one or more components selected from (i) one or more signaling domains; (ii) a transmembrane domain; (iii) a hinge; (iv) IL-15; (v) a T2A sequence; and (vi) a linker.

[0249] The nucleic acid according to Embodiment D13, wherein one or more signaling domains comprise a 4-1BB signaling domain, a CD3ζ signaling domain, or a 4-1BB signaling domain and a CD3ζ signaling domain.

[0250] D15. The nucleic acid according to embodiment D13 or D14, wherein the hinge comprises a CD8α hinge.

[0251] D16. The nucleic acid according to any one of embodiments D1 to D15, wherein the first polynucleotide encodes a first antigen-binding domain comprising a single-stranded variable fragment (ScFv).

[0252] D17.ScFv contains a variable heavy chain (VH) domain and a variable light chain (VL) domain, where, The VH domain comprises a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide, where HVR-H1 comprises the polypeptide of SEQ ID NO: 94, HVR-H2 comprises the polypeptide of SEQ ID NO: 95, and HVR-H3 comprises the polypeptide of SEQ ID NO: 96. The nucleic acid according to Embodiment D16, wherein the VL domain comprises a hypervariable region L1 (HVR-L1) polypeptide, a hypervariable region L2 (HVR-L2) polypeptide, and a hypervariable region L3 (HVR-L3) polypeptide, where HVR-L1 comprises the polypeptide of SEQ ID NO: 98, HVR-L2 comprises the polypeptide of SEQ ID NO: 99, and HVR-L3 comprises the polypeptide of SEQ ID NO: 100.

[0253] The nucleic acid according to embodiment D17, wherein the D18.VH domain contains the polypeptide of sequence number 93 and the VL domain contains the polypeptide of sequence number 97.

[0254] D19. Modified cells transfected or transduced with the nucleic acid described in any one of Embodiments D1 to D19.

[0255] D20. Modified cells according to Embodiment D19, wherein the modified cells are gamma delta T cells.

[0256] D21. A recombinant vector comprising a nucleic acid as described in any one of embodiments D1 to D18.

[0257] D22. A recombinant vector according to embodiment D21, which is a viral vector.

[0258] D23. The recombinant vector according to Embodiment D21, wherein the vector is a non-viral vector.

[0259] A chimeric antigen receptor (CAR) comprising a first antigen-binding domain having binding specificity to E1.TROP2 and a second antigen-binding domain having binding specificity to PD-L1.

[0260] E1.1 The CAR according to Embodiment E1, wherein the first antigen-binding domain specifically binds to the target of TROP2.

[0261] E1.2 The CAR according to Embodiment E1, wherein the first antigen-binding domain specifically binds to two or more targets in TROP2.

[0262] E1.3 The CAR according to any one of embodiments E1 to E1.2, wherein the second antigen-binding domain specifically binds to the target of PD-L1.

[0263] E1.4 The CAR according to any one of embodiments E1 to E1.2, wherein the second antigen-binding domain specifically binds to two or more targets in PD-L1.

[0264] E2. A CAR according to any one of Embodiments E1 to E1.4, wherein the first antigen-binding domain comprises an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0265] E3. A CAR according to any one of Embodiments E1 to E1.4, wherein the first antigen-binding domain comprises an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0266] E4. A CAR according to any one of Embodiments E1 to E1.4, wherein the first antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, and SEQ ID NO: 89.

[0267] E5. The first antigen-binding domain is a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, and 90; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, and 91; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 88, and 92. A CAR according to any one of embodiments E1 to E4, including the CAR described above.

[0268] E6. The CAR according to any one of Embodiments E1 to E5, wherein the second antigen-binding domain comprises an amino acid sequence that is approximately 90% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0269] E7. The CAR according to any one of Embodiments E1 to E5, wherein the second antigen-binding domain comprises an amino acid sequence that is approximately 95% or more identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0270] E8. The CAR according to any one of Embodiments E1 to E5, wherein the second antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0271] E9. The second antigen-binding domain is a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 2, 6, and 10; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 3, 7, and 11; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 4, 8, and 12. A CAR according to any one of embodiments E1 to E8, including the CAR described above.

[0272] E10. A CAR according to any one of Embodiments E1 to E9, comprising a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NOs: 102, 103, 104, 105, 106, and 107.

[0273] E11. A CAR according to any one of Embodiments E1 to E9, comprising a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NOs: 102, 103, 104, 105, 106, and 107.

[0274] E12. A CAR according to any one of embodiments E1 to E9, comprising a polypeptide selected from SEQ ID NOs: 102, 103, 104, 105, 106, and 107.

[0275] E13. The CAR according to any one of Embodiments E1 to E12, wherein the nucleic acid further comprises one or more polynucleotides encoding one or more components selected from (i) one or more signaling domains; (ii) a transmembrane domain; (iii) a hinge; (iv) IL-15; (v) a T2A sequence; and (vi) a linker.

[0276] E14. The CAR according to Embodiment E13, wherein one or more signaling domains include a 4-1BB signaling domain, a CD3ζ signaling domain, or a 4-1BB signaling domain and a CD3ζ signaling domain.

[0277] E15. The CAR according to embodiment E13 or E14, wherein the hinge includes a CD8α hinge.

[0278] E16. A CAR according to any one of embodiments E1 to E15, wherein the first antigen-binding domain comprises a single-stranded variable fragment (ScFv).

[0279] E17.ScFv contains a variable heavy chain (VH) domain and a variable light chain (VL) domain, where, The VH domain comprises a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide, where HVR-H1 comprises the polypeptide of SEQ ID NO: 94, HVR-H2 comprises the polypeptide of SEQ ID NO: 95, and HVR-H3 comprises the polypeptide of SEQ ID NO: 96. The CAR according to Embodiment E16, wherein the VL domain comprises a hypervariable region L1 (HVR-L1) polypeptide, a hypervariable region L2 (HVR-L2) polypeptide, and a hypervariable region L3 (HVR-L3) polypeptide, where HVR-L1 comprises the polypeptide of SEQ ID NO: 98, HVR-L2 comprises the polypeptide of SEQ ID NO: 99, and HVR-L3 comprises the polypeptide of SEQ ID NO: 100.

[0280] E18. The CAR according to Embodiment E17, wherein the VH domain contains the polypeptide of Sequence ID No. 93 and the VL domain contains the polypeptide of Sequence ID No. 97.

[0281] E19. Modified cells containing CAR as described in any one of embodiments E1 to E18.

[0282] E20. Modified cells according to Embodiment E19, wherein the modified cells are gamma delta T cells.

[0283] F1. A pharmaceutical composition comprising modified gamma delta T cells as described in any one of embodiments C1 to C24.

[0284] F2. The pharmaceutical composition according to Embodiment F1, further comprising one or more therapeutic agents selected from antibody immunotherapy, chemotherapeutic agents, biologics, cytokines, or combinations thereof.

[0285] G1. A treatment method comprising administering modified gamma delta T cells described in any one of the embodiments C1 to C24 of the therapeutic dose to a subject in need thereof.

[0286] G2. The treatment method according to Embodiment G1, further comprising co-administering one or more treatment agents selected from antibody immunotherapy, chemotherapeutic agents, biologics, cytokines, or combinations thereof.

[0287] G3. The treatment method according to Embodiment G1 or G2, wherein the subject has cancer.

[0288] G4. A treatment method from any one of embodiments G1 to G3, for a subject having a solid tumor or cancer.

[0289] G5. The treatment method according to any one of Embodiments G1 to G4, wherein the target is one or more of the following: pancreatic cancer, gastric cancer, renal cell carcinoma, lung cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, endometrioid carcinoma, oral squamous cell carcinoma, and papillary thyroid carcinoma.

[0290] A drug that binds to H1.TROP2 or a fragment thereof, a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, and 90; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, and 91; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 88, and 92. A drug containing [this ingredient].

[0291] A drug of Embodiment H1 comprising a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0292] A drug of Embodiment H1 comprising a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0293] A drug of Embodiment H1 comprising a polypeptide selected from SEQ ID NOs: 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, and 89.

[0294] H5. The drug according to any one of embodiments H1 to H4, wherein the drug is an antibody.

[0295] H6. The drug according to any one of embodiments H1 to H4, wherein the drug is a nanobody.

[0296] H7. The drug according to any one of embodiments H1 to H4, wherein the drug is VHH. I1. A drug that binds to PD-L1 or a fragment thereof, a) A hypervariable region H1 (HVR-H1) polypeptide selected from SEQ ID NOs. 2, 6, and 10; b) A hypervariable region H2 (HVR-H2) polypeptide selected from SEQ ID NOs. 3, 7, and 11; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NOs: 4, 8, and 12. A drug containing [this ingredient].

[0297] I2. The agent according to Embodiment I1, comprising a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0298] I3. The agent according to Embodiment I1, comprising a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0299] I4. The agent according to Embodiment I1, comprising a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9.

[0300] I5. The drug according to any one of Embodiments I1 to I4, wherein the drug is an antibody.

[0301] I6. The drug according to any one of embodiments I1 to I4, wherein the drug is a nanobody.

[0302] I7. The drug according to any one of embodiments I1 to I4, wherein the drug is VHH. [Examples]

[0303] Examples The examples described below illustrate specific implementations and are not intended to limit this technology.

[0304] Example 1: Creation of multiple modified subsets of gamma delta T cells This example describes a process for generating multiple highly activated and expanded subsets of gamma delta (γδ) T cells. Typically, γδ T cell therapy products are limited to one γδ subtype: either Vδ1 T cells or Vδ2 T cells, due to production limitations, such as the use of Vδ1 TCR activating agents or phosphoantigens such as zoledronate or BTN3A1 / BTN2A1 agonists that activate only Vδ2 TCRs. Considering the diverse and complementary antitumor activities of both Vδ1 and Vδ2 T cells, the processes described herein have been developed to effectively activate and expand both Vδ1 and Vδ2 T cells together with non-Vδ1 and Vδ2 T cells.

[0305] This embodiment describes a process for generating the modified γδT cells provided herein. Briefly, this process begins with the collection of human apheresis products from a healthy donor, followed by the depletion and cryopreservation of αβT cells using the CliniMACS® TCRα / β product line (Miltenyi).

[0306] For research-scale production, 1 x 10 6Frozen aliquots of αβT-depleted leukocyte apheresis donor samples were thawed and suspended at a rate of 1 million cells per mL in CTS® OpTmizer® T-cell expansion medium (ThermoFisher Scientific) supplemented with 2.5% ICTSR, 1% p / s, 2 mM GLUTAMAX, and 2.5% human AB serum. Soluble anti-CD3 (OKT3) and anti-CD2 agonist (Miltenyi) and human interleukin-15 (IL-15) (R&D) were added at the start of cell culture. In variations of specific workflows, human interleukin-2 (IL-2) may be used in addition to or instead of IL-15 at the start of cell culture. In certain workflow variations, soluble anti-CD3 (OKT3) combined with one or more of NKp44 ligand (PDGF-DD), anti-NKp46, and anti-NKp30; and human interleukin-15 (IL-15) (R&D) and / or human interleukin-2 (IL-2) may be added. Cells were kept cultured, with IL-15 and fresh medium supplied at regular intervals. In certain workflow variations, IL-2 and fresh medium could be supplied to cells at regular intervals until cell harvesting. The process was continuously monitored to determine viability, growth rate, and phenotype. After 14–21 days of culture, expanded γδT cells were phenotypic-classified, and cytotoxicity was determined using co-culture assays with multiple tumor cell lines.

[0307] To generate CARγδT cells, γδT cells were transduced with retroviral vectors encoding TROP2 CAR-IL-15, PD-L1 CAR-IL-15, or TROP2-PD-L1 CAR-IL-15 on day 4 (or days 3-6) of culture. The cell suspension was mixed with CTS complete medium containing the CAR retroviral vector supernatant at MOI=5 and 20 ug / mL of bectofucin-1 (Miltenyi) in a 1:1 ratio, and incubated overnight at 37°C and 5% CO2. The following day, cell culture was continued by adding 4x volume of preheated CST complete medium containing 300 IU / mL of human IL-2. To knock out the TGFβR2 gene in γδT cells, the CRISPR / Cas9 system was delivered on day 6 (or day 4–7) of culture by electroporation on MaxCyte ATX (MaxCyte) as a ribonucleoprotein (RNP) complex containing the Cas9 protein and a single guide RNA targeting TGFβR2.

[0308] The expanded cell composition was determined by flow cytometry analysis for NK cells (CD3-CD56+), αβ T cells (CD3+TCRVαβ+), panγδ T cells (CD3+TCRVγδ+), and Vδ1 (CD3+TCRVδ1+), Vδ2 (CD3+TCRVδ2+), as well as non-Vδ1 and Vδ2 (CD3+Vδ1TCR-Vδ2TCR-αβTCR-) T cell subsets. Activated (CD69, NKG2D) and inhibitory receptor expression, effectors, and memory markers were determined by multichannel flow cytometry analysis. Surface CAR expression was determined by CD34 QBEND / 10 staining and TGFβR2 anti-human TGFβRII staining. All antibodies except TCRVγδ (Miltenyi) and CD34 (Abnova) were purchased from Biolegend.

[0309] Anti-CD2 agonist antibodies enhance the expansion of activated γδ T cells. 1 x 10 6Frozen aliquots of αβT-depleted leukocyte apheresis donor samples were activated with anti-CD3 Ab alone or with both anti-CD3 and anti-CD2 Ab. The addition of anti-CD2 agonist Ab resulted in significantly more innate immune cells by day 21 (Figure 1C). Under both conditions, very pure (approximately 95%) γδ T cells were generated, with residual NK cells and αβ T cells minimized (Figures 1B and 1D). The addition of CD2 agonists also affected the composition of γδ T cells, including subtypes Vδ1, Vδ2, and non-Vδ1 and Vδ2 T cells. Activation with CD2 agonists resulted in comparable levels of Vδ1, more Vδ2, and fewer Vδ1-Vδ2-T cells compared to no CD2 activation (Figures 1B and 1D). However, the innate cytotoxicity of γδT cells, enlarged in the presence or absence of anti-CD2 Ab, was similar to that measured by increasing the E:T ratio from 1:9 to 9:1 in co-cultures with GFP-Luc-expressing pancreatic cancer cell line BxPC3, breast cancer cell line HCC1860, and lung cancer cell line NCI-H1975. Luciferase assays showed comparable tumor-specific lysis profiles in two of the three cell lines tested (Figure 1E).

[0310] The expansion of activated γδT cells possessing IL-2 or IL-15 is similar. One million cryopreserved αβT-depleted PBMCs were thawed, washed, and activated in CTS complete culture medium containing 2.5% human AB serum, 1 ug / mL OKT3 (Biolegend), 0.5 ug / mL anti-CD2 Ab (Miltenyi), and 300 IU / mL human IL-2 (R&D) or 7.5 ng / mL human IL-15 (R&D). The medium or cytokines were replenished every 3 or 4 days throughout the culture. Both cytokines efficiently expanded γδT cells over 21 days of culture and exhibited similar cell viability (Figure 23A), yield (Figure 23B), multiplier of γδT cell expansion (Figure 23C), and purity (Figure 23D).

[0311] Electroporation-mediated delivery of TGFβR2 gRNA / Cas9 RNP for gene knockout (KO) in enlarged γδT cells.

[0312] To disrupt the TGFβR2 gene, three guide RNAs targeting exon 4 of the TGFβR2 gene were identified. Synthetic gRNA (Genscript) was mixed with recombinant Cas9 protein (Genscript) in a 2:1 molar ratio at room temperature to form a ribonucleoprotein (RNP) complex. The RNP was then added to γδT cells washed and resuspended in MaxCyte electroporation (EP) buffer to a final concentration of 3 μM. The mixture of γδT cells (100-200 million cells / mL) and RNP was then transferred to a suitable processing assembly for electroporation in MaxCyte ATX (MaxCyte). After electroporation, the cells were allowed to stand in an incubator for 20 minutes and then returned to culture in preheated medium. After 1 and 3 days, TGFβR2 surface expression was evaluated by flow cytometry using PE anti-TGFβR2 Ab (Biolegend). TGFβR2 expression decreased from approximately 22% in γδT cells electroporated without gRNA to 1-2% in γδT cells with gRNA (Figure 24A). When calculated using the following formula: KO% = (TGFRBR2+% in EP buffer only - TGFRBR2+% in sgRNA) / TGFRBR2+% in EP buffer only * 100%, this represented approximately 90% of the gene knockout efficiency (Figure 24B). Furthermore, TGFβR2 KO γδT cells were highly viable (Figure 24C), with 1 × 10⁶ cells viable on day 0 for EP. 6 They proliferated actively from individual cells (Figure 24D). [Table B-5]

[0313] Characterization of expanded γδT cells and modified γδT cells

[0314] The transduction efficiency of the TROP2-PD-L1 CAR-IL-15 retroviral vector varied in the range of 30%–70%. CRISPR / Cas9-mediated TGFβR2 gene editing was highly efficient, resulting in >90% knockout of TGFβR2 surface expression, as determined by flow cytometry. In TGFβR2 KO, single-donor TROP2 PD-L1 CAR-IL-15γδT cells (PLB-001) were stably transduced to express TROP2-PD-L1 CAR in 61% of cells, while only 3% of cells possessed detectable surface TGFβR2 after TGFβR2-gRNA / Cas9 RNP-mediated gene editing. In contrast, TGFβR2+ cells were detected in 34%–39% of cells that were not gene-edited (Figure 2).

[0315] PLB-001 was highly pure, possessing 94% γδ T cells, with 41.4% exhibiting the central memory (Tcm) phenotype (CD62L+CD45RA-) and 45.5% exhibiting the effector memory (Tem) phenotype (CD62L-CD45RA-). Compared to unmodified γδ T cells, retroviral vector transduction resulted in a slightly reduced Tnaive population from 10% to 4.2%, with no further impact from CRISRP / Cas9 gene editing. Both Tcm and Tem cells possess the potential for adaptive γδ T cell clonal expansion upon receptor occupancy, along with the ability to hom into peripheral tissues and exert effector function. In addition, the enlarged γδT cells were highly active, with the majority expressing the activation markers CD69 (50%) and NKG2D (80%), and a small number of cells expressing the exhaustion markers PD-1 and TIGIT (17%), which were higher than in unmodified γδT cells (4.8%) (Figure 3).

[0316] TROP2-PD-L1 CAR-mediated cytotoxicity Since TROP2 is expressed on a wide range of tumors, tumor cell lines derived from TNBC (HCC70 and HCC1806), mammary adenocarcinoma (SKBR3), lung adenocarcinoma (HCC827 and NCI-1975), pancreatic cancer (BXPC3 and HPAF-II), ovarian cancer (SKOV3), and gastric cancer (NCI-N87), as well as K562 derived from chronic myeloid leukemia that does not express either TROP2 or PD-L1, were purchased from ATCC (Manassas, Virginia). Subsequently, the tumor cell lines were modified to stably express eGFPFfluc by transduction with a lentiviral vector (BPS Bioscience, San Diego, CA). Surface expression levels of TROP2 and PD-L1 were confirmed and quantified by flow cytometry analysis using anti-human TROP2 and anti-human PD-L1 antibodies (Biolegend, San Diego, California) and BD QUANTIBRITE beads (BD Biosciences, San Jose, California).

[0317] The cytotoxicity of PLB-001, TROP2-PD-L1 CAR-IL-15γδT cells (CARγδT), and unmodified γδT cells (NT) against co-cultured GFP-Luc-labeled tumor cell lines was evaluated by luciferase activity assays. Luciferase activity was quantified using the Firefly Luciferase HTS System (Sigma, St. Louis, Missouri). Tumor luciferase activity percentages were calculated for tumor targets only. Tumor-specific lysis was calculated using the formula: 100 × [tumor only - sample) / tumor only. Tumor cell death was also monitored using the BIOTEK CYTATION 5 cell imaging multimode reader (Agilent, Santa Clara, California).

[0318] γδT cells were co-cultured with HCC1806, NCI-H1975, BXPC3, or K562 for 7 days, and after increasing the effector:target (E:T) ratio from 1:9 to 9:1, CARγδT cells showed potent cytotoxicity against TROP2+PD-L1+ tumor cells, achieving approximately 50% tumor lysis (ED50) at E:T ratios of 1:9 (NCI-H1975) or 1:3 (HCC1806 and BXPC3). In the absence of IL-15 supplementation, NTγδT cells also showed spontaneous toxicity against tumor cells, but not as potent as TROP2 PD-L1 CAR-IL-15γδT cells, requiring higher E:T ratios of 1:1 (HCC1806 and NCI-H1975) or 9:1 (BXPC3) to reach 50% tumor lysis. In co-culture with the TROP2-PD-L1-CML cell line K562, CAR-target-independent spontaneous cell death activity was equivalent between CARγδT cells and NTγδT cells, with both reaching 50% tumor lysis at an E:T ratio of 3:1 to 9:1 (Figure 4).

[0319] TGFβR2 knockout significantly improves the antitumor activity of PLB-001. To determine the antitumor activity of PLB-001 against a wide range of tumor types, a diverse array of GFP-Luc-tumor cell lines derived from pancreatic cancer (BXPC3 and HPAF-II), TNBC (HCC70 and HCC1806), breast cancer (SKBR3), lung adenocarcinoma (HCC827 and NCI-1975), gastric cancer (NCI-N87), and ovarian cancer (SKOV3) were tested, all of which express varying degrees of surface TROP2 and PD-L1. Tumor cells were co-cultured with expanded γδT cells from two healthy donors in a 1:2 E:T ratio for up to day 5. As shown in representative fluorescence imaging of pancreatic cell lines BXPC3 and HPAF-II on day 4, GFP-expressing tumor cells were extensively killed by CARγδT cells and almost completely eliminated by PLB-001. Further quantification of tumor cell lysis by luciferase assay on day 5 revealed consistently robust tumor lysis by CARγδ T cells, which was significantly enhanced by PLB-001 (Figure 5).

[0320] To determine PLB-001's resistance to TGFβ1-mediated inhibition, 10 ng / mL of TGFβ1 was added to serum-free co-cultures of γδT cells with GFP-Luc-BXPC3 or HPAF-II pancreatic tumor cells in an E:T ratio of 1:1. Tumor-specific lysis, measured by luciferase assay on day 2, showed that PLB-001 maintained comparable tumor-killing activity regardless of the presence of TGFβ1, whereas CARγδT cells showed significantly reduced tumor-killing activity in the presence of 10 ng / mL of TGFβ1 (Figure 6, Panel A). The enhanced tumor-killing activity of PLB-001 was consistent with the discovery of significantly higher levels of the pro-inflammatory cytokine INFγ, the cytolytic enzyme perforin, and granzyme B in the supernatant of co-cultures of tumor cells with PLB-001 compared to CARγδT cells, as determined by ELISA (R&D) (Figure 6, Panels B-D). The results show that CARγδ T cell activity was significantly negatively affected by TGFβ present in the culture medium of the tumor cell line, while TGFβR2 KO retained PLB-001 activity resistant to TGFβ suppression, which is characteristic of solid tumor TMEs.

[0321] Antitumor activity of PLB-001 in a 3D pancreatic tumor spheroid model Tumor spheroids were seeded in ultra-low adhesion PRIMESURFACE 3D culture round-bottom 96-well plates (S-bio) with 5,000 GFP-Luc-expressing pancreatic tumor cell lines BxPC3 and HPAF-II per well. Once the tumor spheroids were established after 3 days, 5,000 or 15,000 NTγδT cells, TROP2 PD-L1 CARγδT cells, or PLB-001 cells were added for co-culture. Tumor spheroid death was monitored daily, and total green fluorescence intensity was quantified using a BIOTEK CYTATION 5 cell imaging multimode reader (Agilent, Santa Clara, California). NTγδT cells showed limited tumor-killing activity, which was dramatically increased with CARγδT cells and further significantly enhanced with PLB-001. PLB-001 effectively infiltrated tumor spheroids and eliminated all detectable tumor cells over 4 days (Figure 7).

[0322] Sequential tumor-killing activity of PLB-001 after repeated tumor challenge On day 1, multiple plates were seeded with 5,000 GFP-Luc-labeled tumor cell lines NCI-H1975, HPAFII, or BXPC3 per well. On day 0, tumor cells were left untreated (TCs only) or co-cultured with 20,000 PLB-001 or unmodified γδ T cells (NTs). On days 3, 7, and 11, tumor green fluorescence intensity was imaged using BIOTEK CYTATION 5 with dual plates, and tumor-specific killing by luciferase assay, T cell count, and phenotyping were quantified by flow cytometry. Additionally, on days 3 and 7, a new round of tumor challenge was initiated by adding 5,000 fresh tumor cells per well to unused plates.

[0323] PLB-001 demonstrated robust serial killing ability, efficiently eliminating GFP-Luc-labeled tumor cell lines NCI-H1975, HPAFII, or BXPC3 compared to unmodified γδ T cells (Figure 25A), and retained >85% tumor lysis through three tumor challenges (Figure 25B). The sustained antitumor activity of PLB-001 was supported by γδ T cell expansion after tumor antigen binding, reaching 4-30 times in response to different tumor cells by day 11 (Figure 25C). In particular, subsets Vδ1 and Vδ1-Vδ2-T cells showed higher expansion ability than Vδ2 (Figure 25D). Importantly, after three tumor challenges, PLB-001 retained activity and expressed only low levels of exhaustion markers, including PD1+LAG3+ (Figure 25E).

[0324] Tumor homing, invasion, and antitumor activity of PLB-001 in an in vivo tumor xenograft model PLB-001 also demonstrated the ability to hom to, infiltrate, and proliferate tumors in vivo, resulting in significant regulation of tumor growth in a subcutaneous TNBC xenograft model of 6-8 week old NOD / SCID / IL-2γR- / -(NSG) mice (Jackson laboratory). 2 × 10⁶ suspended in PBS 6GFP-Luc-HCC1806 cells were mixed with Matrigel in a 1:1 ratio, totaling 100 μL, and subcutaneously injected into the flanks of mice, with two injections per mouse. Tumors were 50-100 mm in size. 3 After the size was established, six mice were given 10 × 10 in 100 μL of PBS by intravenous injection. 6 Individual cells were treated with PLB-001, while three mice were left untreated. Mouse body weight and tumor size were monitored weekly. The three PLB-001-treated mice were euthanized on day 7, and the remaining animals were euthanized on day 23 to evaluate the tissue distribution and proliferation of PLB-001.

[0325] As shown in Figure 26A, tumors did not grow in PLB-001-treated mice, in contrast to significant tumor growth in untreated animals. PLB-001 was detected in tumors, spleen, blood, and liver (Figures 26B-E) on both days 7 and 23. However, robust expansion of PLB-001 was observed only in tumors, primarily in subsets of Vδ1+ and Vδ1-Vδ2-T cells (Figure 26B), consistent with in vitro findings upon repeated tumor cell challenge. Given the tissue habituation of Vδ2-T cells, the results suggest that PLB-001 may be retained in tissues for long-term tumor surveillance. Furthermore, the absence of PLB-001 expansion in normal tissues also highlights the safety features of γδT cells in the context of allogeneic cell therapy. Since γδT cells do not recognize MHC I, they are unlikely to develop potential graft-versus-host disease. Indeed, the mice maintained normal body weight and did not show clinical signs of GvHD in this study.

[0326] In summary, a process was developed to produce multiple subsets (PLB-001) of γδ T cells derived from healthy donors, expressing the TGFβR2 gene edited by TROP2-PD-L1 CAR IL-15. These cells exhibit robust antitumor activity against TROP2+PD-L1+ tumor cell line cultures and 3D tumor spheroids derived from a wide variety of tumor types, and are resistant to the suppression of TGFβ present in the culture medium. PLB-001 showed continuous tumor-killing activity and robust expansion during in vitro tumor cell challenge, resulting in a significant reduction in tumor burden in in vivo tumor xenograft models.

[0327] Example 2: Generation of anti-PD-L1 VHH (construction, panning, and screening of an immunized alpaca phage library) Adult healthy alpacas were immunized with recombinant human PD-L1-FC protein (GENBANK accession number Q9NZQ7): [ka]

[0328] Animals were boosted four times at two-week intervals. After confirming serum titers against PD-L1, PBMCs were isolated and used to construct a phage display library. Anti-PD-L1 conjugates were isolated using the immunized phage display library with liquid-phase biopanning against biotinylated human PD-L1. Stringency was increased during consecutive rounds by decreasing antigen concentration, increasing the number and duration of washes, and / or changing the selection period. The panning process was repeated 3-4 times until output phage enrichment was identified. Eluten phages were diluted and spread on petri dishes to grow single colonies. Individual phage clones were selected and amplified. 384 single colonies were selected and tested for specificity by ELISA. 264 clones were positive compared to negative controls. After sequencing of the positive clones, three unique sequences were obtained based on amino acid sequences. [Table 1]

[0329] Example 3: Application of PD-L1 conjugates to cell therapy

[0330] When anti-PD-L1 VHH is expressed on the surface of immune cells as a binder in CAR fusion proteins, it can bind to PD-L1-positive tumor cells, activate immune cells, and kill tumor cells.

[0331] A CAR vector containing anti-PD-L1 VHH (ID#PD1,PD2,PD3), CD8α hinge and transmembrane domain, 4-1BB and CD3ζ signaling domains, and human IL-15 isolated by the T2A sequence was constructed by cloning DNA fragments containing all domains into a gamma-retrovirus plasmid (Biovec Pharma, Quebec, Canada). The CD8 hinge amino acid sequence included in this construct is: [ka] The amino acid sequence of the CD8 transmembrane domain included in this construct is: [ka] The amino acid sequence of the 4-1BB signaling domain in this construct is: [ka] The amino acid sequence of the CD3ζ signaling domain in this construct is: [ka] The T2A amino acid sequence included in this construct is: [ka] The amino acid sequence and nucleic acid sequence of human IL-15 are provided below. [ka] [ka]

[0332] Retroviral vectors were prepared by transient transfection of a CAR vector into a 293vecRDF114 packaging cell line (Biovec Pharma, Quebec, Canada) using gene juice (Sigma, St. Louis, Missouri). The retroviral vector supernatant was collected 48 and 72 hours after transfection and stored at -80°C until use.

[0333] Next, PD-L1 CARγδT cells were generated using retroviral vectors encoding CARs derived from different PD-L1 conjugates. Flow cytometry evaluation showed that activated γδT cells from two healthy donors were efficiently transduced using the CAR retroviral vector (Figure 9).

[0334] Example 4: Evaluation of the antitumor activity of PD-L1 CAR-IL-15γδT cells Degranulation and cytokine secretion of PD-L1 CARγδT cells co-cultured with tumor cells. γδT cells modified with different PD-L1 conjugates (CARs) were co-cultured overnight with and without a series of PD-L1-positive tumor cell lines (SKOV3, HCC827, and NCI-H1975). Anti-CD107a-PE antibody (Biolegend, San Diego, California) was added during the first hour, followed by treatment with the secretion inhibitors monensin (2 μM, Biolegend) and brefelzin A (5 μg / ml, Biolegend). The cells were then washed and stained with anti-CD3 BV421 (Biolegend) and anti-CAR CD34 QBEND / 10. CD107a was detected as an indicator of degranulation in CD3+CAR+γδT cells using a flow cytometer (Sony SA3800) (Figure 10, top panel). IFN-γ production by CD3+CAR+γδT cells was analyzed by intracellular staining (Figure 10, bottom panel). Antigen binding to PD-L1-positive tumor cells induces dominant degranulation and upregulation of IFN-γ expression, demonstrating that PD-L1 conjugates are fully functional in activating CARγδ T cells in response to tumors.

[0335] Antitumor cytotoxic effect of PD-L1 CARγδT cells The antitumor cytotoxic activity of PD-L1 CARγδT cells was evaluated against a series of PD-L1-positive cancer cell lines (i.e., SKOV3, HCC827, and NCI-H1975) compared to unmodified γδT cells (NTs) derived from two donors. After co-culturing for 48 hours with each tumor cell line expressing GFP-Luc at indicated E:T ratios of 1:24–4:1, PD-L1 CARγδT cells showed significantly enhanced killing of PD-L1-positive tumor cell lines SKOV3 (Figure 11A), HCC827 (Figure 11B), and NCI-H1975 (Figure 11C) compared to NTs. Unmodified γδT cells also showed innate tumor-killing ability against the ovarian cancer cell line SKOV3 at high E:T ratios (Figure 11A). Antitumor activity of PD-L1 CARγδT cells in a 3D tumor spheroid model

[0336] Tumor spheroids were seeded with 5,000 cells each of GFP-Luc-expressing lung adenocarcinoma cell lines HCC827 and NCI-H1975. When the tumor spheroids were established after 3 days, 10,000, 5,000, or 2,500 PD-L1 CARγδT cells or NTγδT cells were added for co-culture. Tumor spheroids were monitored, and total green fluorescence intensity was quantified daily for up to 5 days using a BIOTEK CYTATION 5 cell imaging multimode reader (Agilent, Santa Clara, California). NTγδT cells showed limited tumor-killing activity, while PD-L1 CARγδT cells effectively infiltrated the tumor spheroids and eliminated all detectable tumor cells over 5 days (Figure 12A, 12B).

[0337] Example 5: Generation of anti-TROP2 VHH (construction, panning, and screening of an immunized alpaca phage library) Adult healthy alpacas were immunized with recombinant human TROP2-FC protein (GENBANK accession number P09758): [ka]

[0338] Animals were immunized three times at 21-day intervals. Cervical blood was collected seven days after each booster immunization and used for serum titer analysis. After the third and fourth immunizations, serum titers significantly increased and showed binding to human and cynomolgus monkey TROP2 even at a 1:256,000 dilution by ELISA, but not to the control human protein, with higher binding of TROP2-CHOK1 cells compared to TROP2-negative CHOK1 control cells.

[0339] After confirming the success of immunization with the TROP2 protein, PBMCs were isolated and measured at 2.42 × 10⁶. 9It was used to construct a phage display library with cfu size. In two parallel discovery campaigns, TROP2 VHH conjugates were isolated from the phage display library using solid-phase panning against human TROP2-His protein and cell-based panning against engineered TROP2-expressing CHOK1 cells. 10 11 Using individual alpaca VHH display phages, 2-3 panning and screenings were performed. Stringency was increased during consecutive rounds by decreasing antigen concentration, increasing the number and duration of washes, and / or changing the selection period. Positively identified phages were diluted and spread on petri dishes to grow single colonies. Individual phage clones were selected and amplified. Panning against human TROP2-His protein yielded 185 positive clones and 16 unique sequences. After three screenings with TROP2 CHOK1 cells, a total of 384 clones were selected, of which 163 were TROP2-specific when tested by ELISA. After sequencing of the positive clones, nine unique sequences were obtained based on their amino acid sequences. Combined, a total of 20 unique sequences were identified.

[0340] To confirm the specificity and functionality of the phages, phages from 20 single colonies were used to stain TROP2-positive cells (TROP2-CHOK1 and MDA-MB-468) and TROP2-negative cells (CHOK1 and 293T) by flow cytometry. As shown in the figures, Figures 13A and 13B show increased binding of TROP2-CHOK1 and MDA-MB-468 cells compared to TROP2-negative CHOK1 cells and 293T control cells, with the exception of TP20, which showed equivalent binding between TROP2-positive and TROP2-negative CHOK1 cells. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0341] Example 6: Application of TROP2 conjugate to cell therapy

[0342] When anti-TROP2 VHH is expressed on the surface of immune cells as a binder in CAR fusion proteins, it can bind to TROP2-positive tumor cells, activate immune cells, and kill tumor cells.

[0343] Twenty anti-TROP2 VHH sequences were individually cloned into a CAR construct containing anti-TROP2 VHH (ID numbers TP1-TP20), CD8α hinge (SEQ ID NO. 137) and transmembrane domain (SEQ ID NO. 138), 4-1BB (SEQ ID NO. 139) and CD3ζ (SEQ ID NO. 140) signaling domains, and human IL-15 (SEQ ID NO. 131), isolated by the T2A sequence (SEQ ID NO. 141) in a gamma-retrovirus plasmid (Biovec pharma, Quebec, Canada). Retroviral vectors were constructed by transient transfection of the CAR vectors into a 293vecRDF114 packaging cell line (Biovec pharma, Quebec, Canada) using gene juice (Sigma, St. Louis, Missouri). Retroviral vector supernatants were collected 48 and 72 hours after transfection and stored at -80°C until use.

[0344] Next, TROP2 CARγδT cells were generated using retroviral vectors encoding CARs derived from different TROP2 conjugates. Flow cytometry evaluation showed that activated γδT cells from two healthy donors were efficiently transduced by the CAR retroviral vector (Figure 14).

[0345] Figures 15A–15C show examples of presented TROP2 CAR conjugates that specifically bind to recombinant human TROP2 protein rather than the homologous human EPCAM protein. The binding affinity (EC50) of TROP2 protein to TP2, TP14, or TP15 VHH conjugate-expressing γδT cells, as determined by flow cytometry, is in the single order of nM, similar to that of γδT cells expressing the TROP2 scFv conjugate adopted from the monoclonal antibody (hRS7) (Figure 16).

[0346] Evaluation of the antitumor activity of TROP2 CARγδT cells Antitumor cell cytotoxicity of TROP2 CARγδT cells The antitumor cytotoxic activity of TROP2 CARγδT cells (pL02) with TP1-TP20 VHH conjugate or hRS7 scFv conjugate was evaluated against a series of TROP2-positive tumor cell lines (i.e., HCC1806, NCI-H1975, HPAF-II, and N87) compared to unmodified γδT cells (NT) derived from two donors. After 48 hours of co-culture with each tumor cell line expressing GFP-Luc at indicated E:T ratios of 5:1 to 1:8, TROP2 CARγδT cells showed significantly enhanced killing compared to NTs of TROP2-positive TNBC cell line HCC1806 (Figure 17A), NSCLC cell line NCI-H1975 (Figure 17B), PDAC cell line HPAF-II (Figure 17C), and gastric cancer N87 cell line (Figure 17D), with the exception of TP16, TP18, and TP19.

[0347] Antitumor activity of TROP2 CARγδT cells in a 3D tumor spheroid model The cytotoxicity of TROP2 CARγδT cells against 3D tumor spheroids was also tested using TROP2-positive cancer cell lines HPAF-II and NCI-H1975. 2,500 GFP-Luc-expressing HPAF-II and NCI-H1975 cells were seeded into tumor spheroids. After 3 days, when the tumor spheroids were established, 1,250 TROP2 CARγδT cells or NTγδT cells were added for co-culture. Tumor spheroids were monitored, and total green fluorescence intensity was quantified daily for up to 7 days using a BIOTEK CYTATION 5 cell imaging multimode reader. NTγδT cells showed limited tumor-killing activity, which was dramatically increased in TROP2 CARγδT cells, except for TP16, TP18, and TP19. TROP2 CARγδT cells effectively infiltrated tumor spheroids and eliminated all detectable tumor cells over 7 days of co-culture (Figures 18A, 18B).

[0348] TROP2 CARγδT cell serial killing ability Subsets of TROP2 CARγδT cells were further tested against repeated challenges (x4) of TROP2-positive tumor cell lines NCI-H1975, N87, and HPAF-II. This assay measures the continuous and sustained tumor-killing capacity of CARγδT cells to achieve persistent tumor control. On day 0, dual plates containing 5,000 GFP-Luc-labeled tumor cells per well were left untreated (TC only) or co-cultured with 20,000 TROP2 CARγδT cells (TP2, TP8, TP9, TP14, TP15, and pL02) or unmodified γδT cells (NT). On days 3, 6, and 9, T cells from one plate were transferred to a new plate seeded with 5,000 fresh tumor cells. Tumor-specific killing was quantified using luciferase assays with dual plates. Compared to NT cells, TROP2 CARγδT cells, with the exception of TP8, maintained significant tumor-killing activity after four tumor antigen challenges, resulting in persistent tumor control. Table 3 below provides the VH, VL, and HVR amino acid sequences of the anti-TROP2 scFv conjugate pL02 used in this example. [Table 3-1] [Table 3-2]

[0349] Example 7: Fabrication and evaluation of bispecific CARs targeting TROP2 and PD-L1 Fabrication and expansion of bispecific CARs

[0350] TROP2 and PD-L1 dual-targeted CARs can be constructed by tandem linking one or more TROP2 conjugate sequences with one or more PD-L1 conjugate sequences in either direction. Table 3 shows examples of dual CARs containing selected TROP2 conjugates TP2, TP9, or TP15 and PD-L1 conjugates PD1 or PD3. Other exemplary constructs include TP2-TP15-PD1, TP15-TP2-PD1, TP2-TP15-PD3, TP15-TP2-PD3, TP2-TP15-PD1-PD3, TP15-TP2-PD1-PD3, TP2-TP15-PD3-PD1, TP15-TP2-PD3-PD1, and other configurations containing TP2, TP9, TP15, PD1, PD3, and / or any other TROP2 and / or PD-L1 VHH polypeptides provided herein in any order. The sequences in Table 4 below were individually cloned into CAR retrovirus (cretroviral) plasmids (Biovec Pharma, Quebec, Canada). Retroviral vectors were constructed by transient transfection of the CAR plasmids into the 293vecRDF114 packaging cell line (Biovec Pharma, Quebec, Canada). Subsequently, TROP2-PD-L1 dual-CAR γδT cells were generated using the retroviral vector supernatant. Flow cytometry evaluation revealed that activated γδT cells from two healthy donors were efficiently transduced with the dual-CAR retroviral vector in the range of 50%–80% (Figure 20A). After 14 days of culture, the expanded γδT cells consisted of 90–95% γδT cells (CD3+γδTCR+) and 1–3% NK cells (CD3-CD56+). The γδ T cells contained approximately 40% Vδ1(CD3+Vδ1TCR+) T cells and 50% Vδ2(CD3+Vδ2TCR+) T cells, respectively (Figure 20B). [Table 4]

[0351] Antitumor cytotoxic effect of TROP2-PD-L1 dual CARγδT cells

[0352] The antitumor cytotoxic activity of TROP2-PD-L1 dual-CARγδT cells was evaluated compared to unmodified γδT cells (NT) in co-culture with TROP2 and PD-L1-positive tumor cell lines (NCI-H1975, HCC827, and SKOV3) with an increased E:T ratio from 1:18 to 4:1. Tumor-specific lysis was determined by luciferase assay 48 hours after co-culture. TROP2-PD-L1 CARγδT cells showed potent cytotoxicity against TROP2+PD-L1+ tumor cells, achieving approximately 50% tumor lysis (ED50) at E:T ratios of 1:1 (in HCC827) or 1:3 (in NCI-H1975 and SKOV3). In the absence of IL-15 supplementation, NTγδT cells also exhibit innate cytotoxicity against tumor cells, though not as potent as TROP2-PD-L1 CARγδT cells, and require a higher E:T ratio of over 4:1 (in NCI-H1975 and SKOV3) to achieve 50% tumor lysis (Figure 21A-C).

[0353] Antitumor activity of TROP2-PD-L1 dual-CARγδT cells in a 3D tumor spheroid model Tumor spheroids were seeded with 5,000 GFP-Luc-expressing NSCLC cells from the NCI-H1975 cell line. When the tumor spheroids were established after 3 days, 2,000, 667, or 222 TROP2-PD-L1 CARγδT cells or NTγδT cells were added for co-culture. Tumor spheroids were monitored, and total green fluorescence intensity was quantified daily for up to 6 days using a BIOTEK CYTATION 5 cell imaging multimode reader (Agilent, Santa Clara, California). NTγδT cells did not show detectable tumor killing, but TROP2-PD-L1 CARγδT cells eliminated all detectable tumor cells by day 5 with an E:T ratio of approximately 1:2.5 (Figure 22A). TROP2-PD-L1 CARγδ T cells expressing the TP15-PD1 conjugate achieved substantial tumor killing with even lower E:T ratios of approximately 1:7.5 and 1:22.5 (Figures 22B and 22C).

[0354] Each patent, patent application, publication, and document referenced herein is incorporated by reference in its entirety. The citation of any patent, patent application, publication, or document does not constitute an endorsement of any of the foregoing as appropriate prior art, nor does it endorse the content or date of any such publication or document. Such citations do not constitute a search for the relevant disclosure. All statements regarding the date or content of any document are based on available information and do not endorse their accuracy or correctness.

[0355] This technology is described with reference to a specific implementation. The terms and expressions used herein to describe this technology are descriptive and not necessarily limiting. Specific modifications made to the disclosed implementation may be considered within the scope of this technology. Certain aspects of the disclosed implementation can be appropriately implemented in or out of the presence of certain elements not specifically disclosed herein.

[0356] Each of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced by any of the other two terms. The terms “a” or “an” may refer to one or more elements that they modify (e.g., “a reagent” may mean one or more reagents) unless the context makes it clear that one, two, or more elements are being described. As used herein, the term “about” refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%; for example, a weight of “about 100 grams” may include weights between 90 grams and 110 grams). When the term “about” is used at the beginning of a list of values, each value is modified (e.g., “about 1, 2, and 3” refers to “about 1, about 2, and about 3”). If an enumeration of values ​​is provided, the enumeration includes all intermediate values ​​and all fractional values ​​(for example, the enumeration of values ​​"80%, 85%, or 90%" includes the intermediate value 86% and the fraction 86.4%). If the term "or greater than" follows an enumeration of values, the term "or greater than" applies to each value listed (for example, the list "80%, 90%, 95%, or greater than" or "80%, 90%, 95%, or greater than" or "80%, 90%, or 95%, or greater than" refers to "80% or greater, 90%, or greater than, or 95%, or greater than"). If an enumeration of values ​​is provided, the list includes all ranges between any two of the listed values ​​(for example, the list "80%, 90%, or 95%" includes the ranges "80% to 90%", "80% to 95%, and "90% to 95%)".

[0357] A specific implementation of this technology is described in the following claims.

Claims

1. Modified gamma delta T cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a first antigen-binding domain having binding specificity to TROP2 and a second antigen-binding domain having binding specificity to PD-L1.

2. The modified gamma delta T cell according to claim 1, wherein the first antigen-binding domain comprises a variable heavy chain domain (VHH) or a single-stranded variable fragment (ScFv) of the heavy chain.

3. The VHH comprises a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide. a) The HVR-H1 polypeptide is selected from SEQ ID NOs: 18, 70, 46, 14, 22, 26, 30, 34, 38, 42, 50, 54, 58, 62, 66, 74, 78, 82, 86, and 90. b) The HVR-H2 polypeptide is selected from SEQ ID NOs: 19, 71, 47, 15, 23, 27, 31, 35, 39, 43, 51, 55, 59, 63, 67, 75, 79, 83, 87, and 91, and c) The HVR-H3 polypeptide is selected from SEQ ID NOs: 20, 72, 48, 16, 24, 28, 32, 36, 40, 44, 52, 56, 60, 64, 68, 76, 80, 83, 88, and 92. The modified gamma delta T cell according to claim 2.

4. The modified gamma delta T cell according to claim 2 or 3, wherein the VHH comprises a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NOs: 17, 69, 45, 13, 21, 25, 29, 33, 37, 41, 49, 53, 57, 61, 65, 73, 77, 81, 85, and 89.

5. The modified gamma delta T cell according to claim 2 or 3, wherein the VHH comprises a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NOs: 17, 69, 45, 13, 21, 25, 29, 33, 37, 41, 49, 53, 57, 61, 65, 73, 77, 81, 85, and 89.

6. The modified gamma delta T cell according to claim 2 or 3, wherein the VHH comprises a polypeptide selected from SEQ ID NO: 17, SEQ ID NO: 69, SEQ ID NO: 45, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, and SEQ ID NO:

89.

7. The ScFv comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, The VH domain comprises a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide, wherein HVR-H1 comprises the polypeptide of SEQ ID NO: 94, HVR-H2 comprises the polypeptide of SEQ ID NO: 95, and HVR-H3 comprises the polypeptide of SEQ ID NO:

96. The VL domain comprises a hypervariable region L1 (HVR-L1) polypeptide, a hypervariable region L2 (HVR-L2) polypeptide, and a hypervariable region L3 (HVR-L3) polypeptide, where HVR-L1 comprises the polypeptide of SEQ ID NO: 98, HVR-L2 comprises the polypeptide of SEQ ID NO: 99, and HVR-L3 comprises the polypeptide of SEQ ID NO:

100. The modified gamma delta T cell according to claim 2.

8. The modified gamma delta T cell according to claim 7, wherein the VH domain comprises the polypeptide of SEQ ID NO: 93 and the VL domain comprises the polypeptide of SEQ ID NO:

97.

9. The second antigen-binding domain comprises a VHH including a hypervariable region H1 (HVR-H1) polypeptide, a hypervariable region H2 (HVR-H2) polypeptide, and a hypervariable region H3 (HVR-H3) polypeptide. a) The HVR-H1 polypeptide is selected from SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10; b) The HVR-H1 polypeptide is selected from SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11; and c) A hypervariable region H3 (HVR-H3) polypeptide selected from SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12, A modified gamma delta T cell according to any one of claims 1 to 8.

10. The modified gamma delta T cell according to claim 9, wherein the VHH comprises a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO:

9.

11. The modified gamma delta T cell according to claim 9, wherein the VHH comprises a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO:

9.

12. The modified gamma delta T cell according to claim 9, wherein the VHH comprises a polypeptide selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO:

9.

13. A modified gamma delta T cell according to any one of claims 1 to 6 and 9 to 12, wherein the CAR comprises a polypeptide that is approximately 90% or more identical to a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO:

107.

14. A modified gamma delta T cell according to any one of claims 1 to 6 and 9 to 12, wherein the CAR comprises a polypeptide that is approximately 95% or more identical to a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO:

107.

15. A modified gamma delta T cell according to any one of claims 1 to 6 and 9 to 12, wherein the CAR comprises a polypeptide selected from SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO:

107.

16. A modified gamma delta T cell according to any one of claims 1 to 15, wherein the CAR further comprises an IL-15 component.

17. The modified gamma delta T cell according to claim 16, wherein the IL-15 component comprises a polypeptide that is approximately 90% or more identical to the polypeptide of Sequence ID No.

131.

18. The modified gamma delta T cell according to claim 16, wherein the IL-15 component comprises the polypeptide of Sequence ID No.

131.

19. A modified gamma delta T cell according to any one of claims 1 to 18, further comprising genome modification.

20. The modified gamma delta T cell according to claim 19, wherein the genome modification is a disrupted gene encoding the TGF-beta2 receptor.

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