Cells that express immunomodulatory molecules and systems for expressing immunomodulatory molecules

Abstract

Disclosed herein are immune cells (i.e., Baize Super cells) engineered to express and incorporate into their cell surface membrane an immune cell activator polypeptide comprising an extracellular targeting domain. Also disclosed are immune cells engineered to secrete one or more polypeptide effector molecules, as well as immune cells engineered to express both molecules. Nucleic acid vectors for expressing these molecules in immune cells are disclosed. Also disclosed are bispecific polypeptides that can be used to specifically bind immune cells expressing an immune cell activator polypeptide to another cell. Also disclosed are systems comprising both immune cells and various bispecific polypeptides that bind to different cell surface proteins on the same or different cellular targets, which can be used, for example, to expand immune cells in vivo and treat various types of tumors.

Description

[Technical Field]

[0001] The subject matter disclosed herein relates to cells expressing immune system regulatory proteins or other effector polypeptides, chimeric immune cell activator polypeptides (ICAPs) (Baize Super cells), and systems used to control the expression of such proteins and polypeptides in those cells. Such systems may include polypeptides having bispecific binding activity, and therefore, when also binding to polypeptide target domains, can activate cells containing vectors for expressing immune system regulatory proteins or other effective polypeptides. [Background technology]

[0002] T cells possessing chimeric antigen receptors (CARs) (CAR-T cells) are being developed as a form of immunotherapy for cancer treatment. Generally, CARs consist of an extracellular domain that binds to an activating ligand, a transmembrane domain that is involved in the formation of immune synapses with "target" cells, and an intracellular domain that responds to the binding of the extracellular domain by activating T cell-associated transcriptional responses.

[0003] Current CAR-T cell-based therapies are ineffective against tumors with heterologous TAA (tumor-associated antigen) expression or novel antigen-deficient variants because the extracellular domain recognizing TAA in CAR cells is monolithic.

[0004] Current CAR-T cell-based therapies rely on in vitro proliferation of CAR-T cells in the patient before treatment.

[0005] Furthermore, there are no readily available methods to monitor the distribution and fate of CAR-T cells in vivo.

[0006] Furthermore, if there is no way to control activated CAR-T cell activity or eliminate undesirable CAR-T cells, CAR-T cells may proliferate continuously and uncontrollably, become activated in response to antigens, and cause fatal on-target off-tumor toxicity, cytokine release syndrome, or neurotoxicity.

[0007] Most CAR extracellular antigen recognition domains are scFv proteins, and it has been shown that two scFv domains can form dimers linked by non-covalent bonds, such as through domain swapping. This type of interaction between adjacent scFv domains strongly enhances sustained signaling in CAR-T cells, leading to uncontrolled activity. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] This specification discloses immune cells that express immune cell activator polypeptides (ICAPs) and are engineered to incorporate them into their cell surface membranes. Also disclosed are immune cells engineered to secrete one or more polypeptide effector molecules, and immune cells engineered to express both molecules.

[0009] Therefore, in one aspect of this disclosure, (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding the amino acid sequence of an immune cell activator polypeptide; (c) Terminator regions effective in terminating transcription in immune cells; Immune cells containing one (or first) nucleic acid vector are provided. These immune cells (d) Promoter regions effective for transcription in immune cells; (e) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (f) A terminator region effective for terminating transcription in immune cells; It can be a cell further comprising a second nucleic acid vector comprising .

[0010] Alternatively, the engineered immune cell can be a cell in which the first nucleic acid vector further comprises a polynucleotide encoding the amino acid sequence of one or more secreted polypeptide effector molecules.

[0011] Another aspect of the disclosure is (a) A labeling domain; (b) A transmembrane domain; and (c) A signaling domain in an immune cell activating factor polypeptide comprising . [[ID=二十]]

[0012] A further aspect of the disclosure is (a) A promoter region effective for transcription in immune cells; (b) A polynucleotide encoding the amino acid sequence of an immune cell activating factor polypeptide; (c) A terminator region effective for terminating transcription in immune cells; A nucleic acid vector comprising .

[0013] Another aspect of the disclosure is (a) A promoter region effective for transcription in immune cells; (b) A polynucleotide encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (c) A terminator region effective for terminating transcription in immune cells; A nucleic acid vector comprising .

[0014] Yet another aspect of the disclosure is (a) A labeling binding domain (L-bd) comprising a single-stranded polypeptide domain that specifically binds to the labeling domain of an immune cell activating factor polypeptide; and (b) A cell surface protein binding domain (CSP-bd) comprising a single-stranded polypeptide domain that specifically binds to a cell surface receptor of a cell, It is a bispecific polypeptide that is a nanobody-targeting and regulatory polypeptide (VHH-TCP) containing [the specified element].

[0015] This disclosure also relates to a kit for in situ production of one or more polypeptide effector molecules proximal to target cells, I. (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding amino acid sequences of immune cell activator polypeptides, including a signaling domain, a transmembrane domain, and a labeling domain; and (c) Terminator regions effective in terminating transcription in immune cells; nucleic acid vectors containing, (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (c) Terminator regions effective in terminating transcription in immune cells; A second nucleic acid vector containing, Immune cells including, II. (a) A label-binding domain (L-bd) containing a single-strand polypeptide domain that specifically binds to the label domain of an immune cell activator polypeptide; (b) A cell surface protein-binding domain (CSP-bd) containing a single-chain polypeptide domain that specifically binds to cell surface receptors, bispecific polypeptides containing The kit, which includes this item, is also described.

[0016] Alternatively, the engineered immune cells may be cells in which the first nucleic acid vector further contains a polynucleotide encoding the amino acid sequence of the secreted effector polypeptide. In such embodiments, the secreted effector polynucleotide may be encoded in a second expression cassette. Such a kit is (a) A label-binding domain (L-bd) containing a single-strand polypeptide domain that specifically binds to the label domain of an immune cell activator polypeptide; and (b) A cell surface protein binding domain (CSP-bd) containing a single-chain polypeptide domain that binds to cell surface receptors on cells. It further comprises a bispecific polypeptide (VHH-TCP) which is a nanobody-targeting and regulatory polypeptide.

[0017] This disclosure also relates to a method for modulating the local immune system environment of tumor cells in a subject, (a) (i) Promoter regions effective for transcription in immune cells; (ii) Polynucleotides encoding the amino acid sequence of an immune cell activator polypeptide; (iii) Terminator regions effective in terminating transcription in immune cells A first nucleic acid vector containing the following: (i) Promoter regions effective for transcription in immune cells; (ii) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (iii) Terminator regions effective in terminating transcription in immune cells Further comprising a second nucleic acid vector containing, A step of administering an effective amount of manipulated immune cells to the target; (b) (i) A label-binding domain (L-bd) containing a single-strand polypeptide domain that specifically binds to the label domain of an immune cell activator polypeptide; and (ii) A cell surface protein-binding domain (CSP-bd) containing a single-strand polypeptide domain that specifically binds to cell surface receptors of lymphocytes. A step of administering an effective amount of a first bispecific polypeptide, including the above, simultaneously or sequentially to the target; (c) (i) A label-binding domain (L-bd) containing a single-strand polypeptide domain that specifically binds to the label domain of an immune cell activator polypeptide; and (ii) A cell surface protein binding domain (CSP-bd) containing a single-chain polypeptide domain that specifically binds to the cell surface proteins of tumor cells. A step of administering an effective amount of a second bispecific polypeptide, including the above. We also provide methods that include this.

[0018] The step of measuring the amount of manipulated immune cells within the subject can be performed between steps b and c.

[0019] Alternatively, methods for modulating the local immune system environment of tumor cells in a target include: (a) A step of growing the target transformed T cells in vitro to obtain the proliferated T cells, wherein the T cells (i) Promoter regions effective for transcription in immune cells; (ii) Polynucleotides encoding the amino acid sequence of an immune cell activator polypeptide; and (iii) Terminator regions effective in terminating transcription in immune cells, A first nucleic acid vector containing a nucleic acid vector; and (i) Promoter regions effective for transcription in immune cells; (ii) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; and (iii) Terminator regions effective in terminating transcription in immune cells, A step comprising a second nucleic acid vector containing; and A step of administering the proliferated T cells; Furthermore (b) A step of targeting an effective amount to activate proliferated T cells and express secreted polypeptide effector molecules of VHH-TCP, which include L-bd with a determined amino acid sequence that specifically binds to a labeled domain expressed by the proliferated T cells and CSP-bd that specifically binds to the cell surface protein of tumor cells. It may include.

[0020] This specification concludes with claims that specifically point to and expressly assert the subject matter described herein, which is considered to be better understood from the following description of specific embodiments in conjunction with the accompanying drawings, in which similar reference numbers identify the same elements: [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows exemplary effector cells described herein, such as "Baize Super cells," which express immunomodulatory effector polypeptides and secrete immunomodulatory effector molecules, and also shows anti-PD1-VHH nanobodies. [Figure 2] This figure shows a nanobody-targeting and regulatory polypeptide (VHH-TCP), with a labeling domain for immune cell activator proteins (labeled-VHH) and a domain that binds to cell surface proteins, in this example to CD19 ligand on B cells (CD19-VHH). Additional domains for activating Fc-mediated immune responses (hFc-VHH), binding to FITC fluorescent dyes (FITC-VHH), and binding to serum albumin (albumin-VHH) are also shown. [Figure 3A]This figure shows exemplary expression vector maps for expressing ICAP and effector proteins in immune cell-type host cells. In Figure 3A, the effector protein anti-PD-1-VHH-Fc(EQ) is expressed from the structural gene of the expression construct within the vector pS338B-1182-Fc(EQ). In Figure 3B, ICAP, which has a labeled polypeptide domain, a CD8 hinge domain, a CD28 transmembrane (TM) domain, a CD28 intracellular signaling domain, and a CD3z domain, is expressed from the structural gene within the expression construct pNB338B-ICAP-VHH. [Figure 3B] This figure shows exemplary expression vector maps for expressing ICAP and effector proteins in immune cell-type host cells. In Figure 3A, the effector protein anti-PD-1-VHH-Fc(EQ) is expressed from the structural gene of the expression construct within the vector pS338B-1182-Fc(EQ). In Figure 3B, ICAP, which has a labeled polypeptide domain, a CD8 hinge domain, a CD28 transmembrane (TM) domain, a CD28 intracellular signaling domain, and a CD3z domain, is expressed from the structural gene within the expression construct pNB338B-ICAP-VHH. [Figure 4] Figure 4A shows the flow binding affinity of M2339(VHH) to mesoserine (MSLN). Figure 4B shows the flow binding affinity of B029(VHH) to mesoserine (MSLN). Figure 4C shows the flow binding affinity of E454(VHH) to EGFR. [Figure 5] This figure shows the binding kinetics of M2339VHH-6his to various mesoserine ECD domains as determined by surface plasmon resonance (SPR). [Figure 6]This figure shows schematic diagrams of various M-ICAP (derived from the mesoserine II+III region) vectors. 19R73 is the standard CD19CAR-T type (positive control), while the others are M-ICAP vectors. The intracellular regions of these vectors are the same, all containing 4-BB and CD3ζ, but the extracellular regions differ. M-ICAP does not contain the 6-His tag. His-1 / 2-M-ICAP: The 6-His tag is located at the N-terminus or C-terminus of M-ICAP. SP3-His-M-ICAP and SP5His-M-ICAP: The signal peptide is SP3 or SP5 selected from the human protein database. SP3 (Signal Peptide 3): MKHLWFFLLLVAAPRWVLS (Sequence ID 1); SP5 (Signal Peptide 5): MTRLTVRALLAGLLASSRA (Sequence ID 2); [Figure 7] This figure shows the FACS results of M-ICAP vectors transfected into 293T cells. Figure 7A shows the positivity rates of various M-ICAP vectors transfected into 293T cells. Figure 7B shows a dot plot of the FACS results. [Figure 8] This figure shows the expression of M-ICAP and the structure of M-ICAP-T cells. Figure 8A is a schematic diagram showing the structure of M-ICAP-T cells. Figure 8B provides schematics of M-ICAP, SP3-M-ICAP, and SP5-M-ICAP expression vectors. Figures 8C and 8D show data on the positivity rates of M-ICAP, SP3-M-ICAP, and SP5-M-ICAP T cells 8 and 13 days after transfection (Figure 8C: activated by M2339 + anti-CD28 or anti-His + anti-CD28, respectively; Figure 8D: activated by M2339 + anti-CD28). Abbreviations: M-ICAP-Mesoserine II+III derived peptide; SP-Mesoserine-Endogenous signal peptide of mesoserine; SP3 (Signal Peptide 3): MKHLWFFLLLVAAPRWVLS (Sequence ID 1); SP5 (Signal Peptide 5): MTRLTVRALLAGLLASSRA (Sequence ID 2); M2339, anti-M-ICAP-VHH-Fc clone M2339; anti-CD28-anti-CD28 mAb; anti-His-anti-HismAb. [Figure 9] This figure shows typical preparation and quality validation of ICAP-T cells. Figure 9A provides a schematic of M-ICAP, M-ICAP-28, and M-ICAP-28BB expression vectors. Figure 9B shows a comparison of ICAP-T amplification (peripheral blood monocytes, PBMCs) obtained by activation of different TCPs or antibodies. Figure 9C shows the growth of ICAP-T cells during preparation from PBMCs. Figure 9D shows the percentage of ICAP-positive ICAP-T cell products. Figure 9E shows the percentage of CD4 / CD8-positive ICAP-T cell products in CD3-positive cells. Figure 9E shows the percentage of Tem / Tcm-positive ICAP-T cell products in CD3-positive cells. [Figure 10] This figure shows the binding affinity of BCMA-TCP as measured by FACS. Figure 10A shows the FACS binding curves of three BCMA-TCPs to cells of an MSLN-overexpressing cell line. Figure 10B shows the FACS binding curves of three BCMA-TCPs to cells of a BCMA-overexpressing cell line. [Figure 11] This figure shows the plasma stability of anti-BCMA TCP. [Figure 12] This figure shows the binding affinity of TCP011-P to two different cell types as measured by FACS. Figure 12A shows the FACS binding curve of TCP011-P to cells in a CD19-overexpressing cell line. Figure 12B shows the FACS binding curve of TCP011-P to cells in an MSLN-overexpressing cell line. [Figure 13] This figure shows the binding affinity of TCP021-P to two different cell types as measured by FACS. Figure 13A shows the FACS binding curve of TCP021-P to EGFR-overexpressing cells. Figure 13B shows the FACS binding curve of TCP021-P to MSLN-overexpressing cells. [Figure 14]This figure shows the in vitro amplification of M-ICAP-T by TCP against target cells. Figures 14A and 14B show the number of T / Daudi cells after co-culturing M-ICAP-transfected T cells and Daudi cells with TCP for 4 days. Figures 14C and 14D show the number of T / Daudi cells after co-culturing M-ICAP-transfected T cells and Daudi cells treated with mitomycin C (MMC) with TCP for 4 days. [Figure 15] This figure shows the TCP dose-dependent cytotoxic effect of M-ICAP-T on RPMI-8226 cells. Figure 15A shows a schematic of a cell lysis assay in suspension cells. Figure 15B shows the dose-dependent cell lysis release ratio of M-ICAP-T combined with TCP001-C for RPMI-8226 cells at three different E:T ratios. Figures 15C-15E show the cell lysis analysis curves for different E:T ratios. [Figure 16] This figure compares the cytotoxicity and IFNγ secretion of ICAP / CAR-T cells combined with different TCPs against RPMI-8226 / L363 cells. Figures 16A and 16B show a comparison of the cytotoxic effects of ICAP / CAR-T cells against L363 cells combined with different TCP concentrations of 0.5 μg / ml (A) or 0.2 μg / ml (B). Figures 16C and 16D show IFNγ secretion of ICAP / CAR-T cells combined with different TCPs against RPMI-8226 (C) or L363 (D) cells. [Figure 17] This figure shows the cytolytic effect of ICAP combined with TCP (which binds to EGFR) on FaDu / SK-OV3 cells. Figure 17A shows the cytolysis of FaDu cells by ICAP / CAR-T cells combined with different TCPs. Figure 17B shows the cytolysis of SK-OV3 cells by ICAP / CAR-T cells combined with different TCPs. [Figure 18]This figure shows the IFN-γ release and cytolytic effects of ICAP-T cells with TCP against Daudi cells. Figure 18A shows IFN-γ release from ICAP / CAR-T cells with different TCPs against Daudi cells. Figure 18B shows cytolysis of Daudi cells by ICAP / CAR-T cells combined with different TCPs. [Figure 19] This figure shows that M-ICAP-T cells possess the ability to secrete antibodies, and that the positive rate is not affected. Figure 19A shows a comparison of the positive rates of secretory M-ICAP-T cells. Human naive T cells were simultaneously transfected with M3 CAR, and plasmids of antibodies such as anti-PD-1, anti-TGFβ, and anti-PD-L1 were secreted. After 13 days, there was little difference among the four experimental groups, with a positive rate of approximately 60-70%. Anti-PD-1, anti-TGFβ, and anti-PD-L1 antibodies were also sufficiently secreted from M-ICAP-T cells. These data indicate that the type and level of antibody secretion have little effect on the positive conversion of M-ICAP-T cells. Both VHH and scFv were sufficiently secreted from M-ICAP-T cells and can be detected by ELISA. [Figure 20] This figure shows that anti-PD-1-M-ICAP-T cells can secrete anti-PD-1 VHH, thereby blocking the surface PD-1 protein. Cells were stimulated for 48 hours with 5 μg / ml M2339-IgG4 or an IgG4 control. Only the detection of surface PD1 in the anti-PD-1VHHM-ICAP-T cell population was blocked by a commercially available PD-1 mAb. [Figure 21] This figure shows that anti-TGFβ scFv secreted by M-ICAP-T cells binds to TGFβRII. The TGFβ ligand binds to TGFβRII and stimulates luciferase signaling. Anti-TGFβ scFv secreted by M-ICAP-T cells can also bind to TGFβRII on 293T cells and block the expression of the luciferase reporter. CAR-T-10C, 10B, and 01A are anti-TGFβ M-ICAP-T cells prepared from different donors. [Figure 22]This figure shows the change in body weight of L363-PDL1 orthotopic tumors in NPSG mice during an in vivo efficacy study of M-ICAP-T cells combined with TCP001-C. [Figure 23] This figure shows the change in tumor volume of L363-PDL1 orthotopic tumors in NPSG mice during an in vivo efficacy study of M-ICAP-T cells combined with TCP001-C. [Figure 24] This figure shows the analysis of anti-PD-1VHH and TCP001-C concentrations in whole blood of mice. Figure 24A shows the analysis of serum anti-PD-1VHH levels. Figure 24B shows the analysis of serum TCP001-C levels. Abbreviations: D15-24h, tail vein blood collection on day 15, 24 hours after TCP001-C injection on day 14; D22-48h, tail vein blood collection on day 22, 48 hours after TCP001-C injection on day 20. The promoter in each of the vectors shown is the EF1a promoter, and the SV40 polyadenylation signal is used for transcription termination in both vectors. Both expression constructs contain 5' and 3' ITR sequences. [Figure 25] This figure shows the binding of anti-MSLN-1444VHH (1444(VHH)) to HEK293T-MSLN cells as analyzed by FACS. [Figure 26] This figure shows the expression of the fusion polypeptide BCMA ICAP BCMAmut1 with anti-MSLN-1444VHH. [Figure 27] This figure shows the SPR kinetics of BCMA ICAP BCMAmut1, which binds to various anti-BCMA VHHs. [Figure 28]This figure shows the in vitro activation and amplification of BCMAmut1-MSLN-1444 CAR-T. Figure 28A shows a schematic diagram of the BCMAmut1-MSLN-1444 vector. Figure 28B shows the amplification of BCMAmut1-MSLN-1444 CAR-T stimulated with anti-BCMAmut1 VHH36# or anti-MSLN in donor 1. Figure 28C shows the amplification of BCMAmut1-MSLN-1444 CAR-T stimulated with anti-BCMAmut1 VHH36# or anti-MSLN in donor 2. [Figure 29] This figure shows dot plots of FACS results for amplification of BCMAmut1-MSLN-1444CAR-T stimulated with anti-BCMAmut1 VHH 36# or anti-MSLN in two donors. [Figure 30] This figure shows the anti-BCMAmuc1VHH 36# specific activation and amplification of MSLN-1444CAR-T. Figure 30A shows a schematic diagram of the MSLN-1444 vector. Figure 30B shows the amplification of MSLN-1444 CAR T stimulated with anti-BCMAmuc1 VHH 36# or the antigen MSLN in donor 1. Figure 30C shows the amplification of MSLN-1444 CAR T stimulated with anti-BCMAmuc1 VHH 36# or anti-MSLN in donor 2. [Modes for carrying out the invention]

[0022] Chimeric antigen receptor T cell (CAR-T) treatment technology is a field of cancer immunotherapy. CAR-T technology uses genetic engineering techniques to splice an antibody variable region gene sequence, for example, containing at least a portion of the gene encoding the CDR portion of an antibody, onto the intracellular region of a T lymphocyte immune receptor. The splice construct is then introduced into T cells via retroviral or lentiviral vectors, transposons, or transfection. The expression cassette or mRNA is transduced into the lymphocytes, causing them to express a fusion protein on the cell surface, enabling T lymphocytes to recognize specific antigens in an MHC-independent manner, thereby enhancing their ability to recognize and kill tumors.

[0023] The structure of chimeric antigen receptors (CARs) was proposed in 1989 by the Eshhar research team in Israel. Since then, T cells exhibiting CAR-structured cell surface proteins have been shown to have favorable effects in tumor immunotherapy.

[0024] First-generation CAR receptors contain a single-chain variable region fragment (scFv), and intracellular activation signals are transmitted via the CD3ζ (CD3z) signaling chain. However, because first-generation CAR receptors lack domains that provide T cell costimulatory signals, CAR-T cells exert only transient effects, resulting in shorter cell survival in the body and reduced cytokine secretion. Second-generation CAR receptors incorporate intracellular domains of costimulatory signaling molecules, including, for example, CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory factor (ICOS), DNAX-activated protein 10 (DAP10), and other domains that enhance T cell proliferation and cytokine secretion. Increased production of IL-2, IFN-γ, and GM-CSF disrupts immunosuppression in the tumor microenvironment, leading to conditions such as AICD (activation-induced cell death (AICD)).

[0025] Third-generation CAR receptors recombine secondary costimulatory molecules such as 4-1BB between the costimulatory structure CD28 and the ITAM signaling chain, thereby generating a 3-signal CAR receptor.

[0026] Manipulated CAR-T cells exhibit better effector function and survival in vivo. The CAR structure currently commonly used in therapeutics is the second-generation CAR receptor, whose structure can be divided into four parts: the antibody single-chain variable region (scFv), the hinge region, the transmembrane region, and the intracellular stimulus signaling polypeptide. The CAR hinge region structure contributes to the formation of correct conformation and dimerization. The length and amino acid sequence characteristics of the hinge region contribute to determining the spatial conformation of the CAR and also affect the CAR's ability to bind to tumor cell surface antigens.

[0027] Malignant lymphoma is divided into two categories: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Hodgkin lymphoma accounts for 10-15% of lymphomas, but non-Hodgkin lymphoma is the most rapidly growing malignant tumor among those who develop the disease. According to WHO statistics, there are currently about 350,000 new NHL cases worldwide each year, and more than 200,000 deaths. B-cell lymphoma can be found in both Hodgkin lymphoma and non-Hodgkin lymphoma. Currently, clinical treatment for lymphoma includes cytotoxic drugs such as glucocorticoids and alkylating agents, and targeted drugs based on specific molecular targets (such as rituximab), and combination chemotherapy based on targeted drugs significantly improves patient response, clinical remission rates, and cure rates. However, there are a large number of lymphoma patients who are unresponsive or have an insufficient response and are "actually" refractory patients. Several new treatments (such as cellular immunotherapy) have reduced the severity of partially relapsed or refractory lymphoma in patients and extended their survival. Many types of CAR-T therapy are currently under development for hematological malignancies, including therapies using anti-CD19, anti-CD20, anti-kappa light chain, anti-CD22, anti-CD23, anti-CD30, anti-CD70, and other antibodies to construct CAR-modified T cells. Antitumor trials have been conducted, in which these anti-CD19 and anti-CD20 monoclonal antibodies were the most commonly used.

[0028] Selecting the appropriate tumor antigen as a target is key to designing safe and effective CAR-T cells. CD19 is a potential target for treating B-lineage tumors and a hot spot in CAR-T research because it is expressed only in normal and malignant B cells at various stages of differentiation, and not in other non-B cells (such as hematopoietic stem cells). Therefore, CD19 CAR-Ts are widely used for malignancies such as acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL, and multiple myeloma (MM). CD19 CAR-Ts are also being used in clinical trials for treating B-cell lymphomas.

[0029] PD-1 (Programmed Cell Death 1, Reprogrammed Cell Death Receptor 1) is a member of the regulatory T cell CD28 family and belongs to the immunoglobulin receptor superfamily. PD-1 and its ligands, PD-L1 / PD-L2, play crucial roles in T cell co-suppression and impairment. Their interaction inhibits the proliferation and cytokine secretion of costimulated T cells. Expression of the anti-apoptotic molecule BCL-xl impairs the function of tumor-specific T cells, which prevents some tumor patients from completely eliminating their tumors. Anti-PD-1 antibodies compete with ligands PD-L1 / PD-L2 for binding to the PD-1 molecule on the surface of tumor-specific T cells, thereby inhibiting the formation of a PD-1-PD-L1 / PD-L2 complex. This, in turn, overcomes the inhibition of the immune microenvironment caused by the formation of a PD-1-PD-L1 / PD-L2 complex.

[0030] The currently available anti-PD-1 antibodies are nivolumab and pizilizumab. These two monoclonal antibodies have been shown to have excellent clinical efficacy in solid tumors such as melanoma, colorectal cancer, prostate cancer, non-small cell lung cancer, and renal cell carcinoma. Recent clinical studies have confirmed that PD-1 antibodies can be used to treat lymphoma. However, anti-PD-1 antibodies still have several unavoidable problems in clinical application. On the one hand, because anti-PD-1 monoclonal antibodies are administered intravenously, most patients receiving PD-1 antibody blockers experience varying degrees of drug side effects. Furthermore, in vitro production of anti-PD-1 monoclonal antibodies involves complex production preparation and purification processes, which are costly and expensive.

[0031] In summary, CAR-T cells have the ability to kill tumor cells and effectively invade tumor tissue, but these activities are easily inhibited in the tumor microenvironment, and PD-1 antibodies can reactivate the antitumor activity of T cells. However, conventional high molecular weight antibodies or their large fragments have insufficient ability to penetrate solid tumors, systemic drugs have significant toxic side effects, and the cost of the drugs is high.

[0032] Therefore, a solution to this problem is disclosed herein, in which an anti-PD-1 antibody can be effectively expressed by maintaining the toxic toxicity of CAR-carrying immune cells (e.g., CAR-T cells) and the PD-1 antibody, and the PD-1 antibody is expressed at high levels within or near the tumor by CAR-carrying cells. This activity is expected to enhance the tumor-killing effect of CAR-carrying cells while reducing treatment costs.

[0033] This specification discloses a system that is similar to CAR-T cells but possesses several features of more generalized properties. Furthermore, by including an extracellular peptide molecule (possibly synthetic and naturally occurring amino acid sequences) with bispecific binding activity that binds to both CAR-bearing effector cells and target cells possessing cell surface antigens, the activity level of CAR-bearing effector cells can be regulated by controlling the amount of ICAP available to bind to the CARs. Applying such a system can solve the problem of high sustained activity exhibited by conventional CAR-T cells.

[0034] Some of the terms related to this disclosure are explained below.

[0035] In this disclosure, the term “expression cassette” refers to the entire set of elements required for gene expression, including the promoter, coding sequence, and polyA tailing signal sequence.

[0036] The term "coding sequence" is defined herein as a portion of the nucleic acid sequence that encodes the amino acid sequence of a polypeptide product (e.g., a CAR, a single-chain antibody, or its domain). The boundaries of a coding sequence are typically determined by the ribosome binding site immediately upstream of the 5' open reading frame at the 5' end of the encoded mRNA (in the case of prokaryotic cells) and the transcription termination sequence immediately downstream of the 3' open reading frame at the 3' end of the encoded mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0037] The term "Fc" (crystallizable fragment) refers to a peptide that is part of a mammalian antibody, located at the end of the handle of the "Y" structure of the antibody molecule, containing the CH2 and CH3 domains of the antibody's heavy chain constant region, and is also the site of many molecular and cellular interactions that provide some of the biological effects of mammalian antibodies.

[0038] The term "costimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that binds to costimulatory molecule receptors on Th cells, generating costimulatory signals. Lymphocyte proliferation requires not only antigen binding but also costimulatory molecule signals. Costimulatory signals are transmitted to T cells primarily by binding to the costimulatory molecule CD80 on the surface of antigen-presenting cells, while CD86 binds to the CD28 molecule on the surface of T cells. B cells receive costimulatory signals that can pass through common pathogen components such as LPS, complement components, or the activated antigen-specific Th cell surface protein CD40L.

[0039] The term "linker" refers to a polypeptide fragment that links different proteins or polypeptides together for the purpose of maintaining the spatial relationship between linked proteins or polypeptides, for example, by mitigating steric inhibition of ligand binding, thereby maintaining the function or activity of the protein or polypeptide. Exemplary linkers include glycine and / or serine, as well as linkers containing, for example, the furin 2A peptide.

[0040] The term "specifically binds" refers to a reaction between a binding protein and a ligand, such as between an antibody or antigen-binding fragment and the antigen it is directed towards. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) has an antibody-antigen affinity of approximately 10 -5 Coupling constants Kd less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 This means that it is characterized by being M or less. "Specifically recognized" or "specific recognition" have the same meaning.

[0041] The term “pharmaceutically acceptable excipients” refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredients, as is well known in the art (e.g., Remington's Pharmaceutical Sciences, editor Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995, incorporated herein by reference in whole and for all purposes), and includes, but is not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, e.g., Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0042] The term "effective dose" refers to a dose that can achieve the treatment, prevention, relief, and / or relief of the disease or condition described herein in the subject.

[0043] The term “disease and / or condition” refers to the physical condition of the subject relating to the disease and / or condition described herein.

[0044] The terms “subject” or “patient” may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cattle, horse, etc., that receives a pharmaceutical composition of the present invention to treat, prevent, improve and / or alleviate a disease or condition of the present invention.

[0045] As used herein, “chimeric antigen receptor” (CAR) is an artificially engineered protein that binds to a specific molecule, such as a tumor cell surface antigen, and stimulates the proliferation program in immune cell-type effector cells. A CAR typically comprises, from amino-terminus to carboxy-terminus, an arbitrary signal peptide (which may be removed during the process of localization of the CAR on the host cell membrane); a polypeptide that specifically binds to another protein, such as the antigen-binding region of a single-chain antibody ("labeled domain"); an optional (but typically present) hinge region; a transmembrane region; and an intracellular signaling region (see, e.g., Figure 1). The labeled domain polypeptide may be derived from a native polypeptide or a synthetic polypeptide.

[0046] In this application, “VHH domain” may refer to the variable domain of a single heavy chain antibody (“VHH antibody”), such as a camelid antibody. A “single-chain antibody” (SCA) is a single-chain polypeptide that typically includes several relatively conserved domains that associate together when the polypeptide folds to form a framework region (FR region), and a variable region that binds together to form a variable antigen-binding domain. Therefore, a VHH antibody is a type of SCA. According to this terminology, the variable domain present in naturally occurring single-chain heavy chain antibodies is also referred to herein as the “VHH domain” to distinguish it from the heavy chain variable domain (referred herein to as the “VH domain”) and the light chain variable domain (referred herein to as the “VL domain”) present in conventional quadruple-chain antibodies.

[0047] The isolated single variable domain polypeptides are preferably polypeptides that possess the full antigen-binding ability of their homologous SCAs and are stable in aqueous solution.

[0048] Stable antigen-binding single-chain polypeptides containing one or more domains (either FR or variable region origin) derived from or similar to the domains of mammalian antibodies, such as the VH domain, are also included in the term "single-chain antibody" as used herein.

[0049] The term "nanobody" may include an SCA or VHH antibody, or one or more domains thereof, but the word is more typically used to describe an engineered polypeptide that includes one or more VHH domains and optionally one or more further FR domains, and additionally or alternatively includes additional stable domains having some further biological activity, such as binding to a fluorescent dye or binding and activation of an extracellular receptor.

[0050] In this specification, a novel cell therapy product, an engineered immune effector cell, is disclosed, which may be a cell such as a so-called "Baize Super cell" containing a chimeric receptor that can be induced to express secreted proteins in a controllable manner in situ.

[0051] In some embodiments, engineered immune effector cells constitutively express high levels of effector polypeptides, such as single-chain anti-PD-1 antibody (VHH-PD-1). In some such embodiments, cell surface-associated antigens of cells are activated by binding to the "labeling domain" of immune effector cells, which are T cells. The proliferation of T cells provides a very large number of T cells that constitutively secrete effector polypeptides. When antigens on the surface of tumor cells bind to the labeling domain, the immunomodulatory effector polypeptides may be polypeptides that, by being constitutively expressed and secreted proximal to tumor cells, mitigate or evade immune tolerance induced, for example, by PD-1:PD-L1 / L2 complex formation.

[0052] Additionally or alternatively, the engineered effector cells disclosed herein may be engineered to include a nucleic acid vector comprising a coding sequence construct encoding one or more “effector polypeptides” expressed under the control of an activatable promoter in immune cells, and further comprising a transcription termination sequence activatable in immune cells. The promoter may be a constitutive promoter such as the EF1a promoter or the CMV promoter.

[0053] Nucleic acid vectors can be retroviral vectors or lentiviral vectors. Nucleic acid vectors can be DNA or RNA vectors. Vectors can contain PiggyBac (PB) transposons or SleepingBeauty (SB) transposons or parts thereof. Vectors can typically contain transposon-specific reverse-terminal repeat sequences located at both ends of a transposon-based vector.

[0054] The engineered effector cells disclosed herein may have either or both an expression cassette encoding ICAP and / or an expression cassette encoding one or more effector polypeptides incorporated into the nuclear genome of the effector cell.

[0055] The proteins secreted by effector cells may be immunostimulant proteins, such as polypeptides that specifically bind to 4-1BB or OX40, or immunosuppressive proteins (for example, to treat allergic responses or arthritis conditions), such as polypeptides that specifically bind to TNF-α or IL-6.

[0056] Preferred proteins secreted by effector cells are antibodies or fragments thereof, or polypeptides that are single-chain, single-domain polypeptides, such as VHH nanobodies or scFv proteins. One class of proteins that can be secreted is immune checkpoint receptor antagonist or agonist antibodies, with or without an Fc domain. However, other proteins, such as cytokines or other immunomodulatory proteins, can be expressed and secreted by engineered effector cells. For example, antibodies against PDL1, CTLA-4, CD-40, LAG-3, TIM-3, BTLA, CD160, 2B4, CD40, 4-1BB, GITR, OX-40, CD27, HVEM, or LIGHT, the antigen-binding portion of an antibody, or single-chain antibodies, such as VHH nanobodies, can be expressed and secreted from effector cells. Examples of cytokines secreted from effector cells include TGF-β, VEGF, TNF-α, CCR5, CCR7, IL-2, IL-7, IL-15, and IL-17. The engineered effector cells of the present invention can express and secrete two or more different types of effector polypeptides, such as different antibodies, cytokines, or combinations thereof. For example, engineered effector cells can secrete anti-PDL1 antibody and anti-CTLA-4 antibody, or anti-PDL1 antibody and VEGF antibody.

[0057] An example of a secreted effector protein is the anti-PD-1VHH antibody (1182) having the amino acid sequence QVQLVESGGGLVQAGGSLRLSCAASGDTSFISAAGWYRQAPGKERELVAAITNTGITYYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCNAGAPPPGGLGYDESDYWGQGTQTV (SEQ ID NO: 3).

[0058] The host immune cells manipulated may include various T cells, CIKs (cytokine-induced killer cells), DC-CIKs (dendritic cell / CIKs), NKs (natural killer cells), NKTs (natural killer T cells), stem cells, TILs (tumor-infiltrating lymphocytes), macrophages, and other immune cells. Host immune cells are typically the autologous cells of the target being treated for a disease.

[0059] In some embodiments, engineered immune cells are transformed by a vector comprising a coding sequence construct having at least three structural components: a first domain containing an intracellular signaling domain that activates the transcription program in “activated” T cells, e.g., a polynucleotide encoding the CD3ε (CD3e) or CD3ζ (CD3z) domain of a T cell surface glycoprotein; a second polynucleotide encoding a domain containing a transmembrane domain (and optionally a spacer peptide), e.g., a domain from the CD28 protein; and a third polynucleotide encoding a domain that is a “labeled” polypeptide, the specific binding of which by another polypeptide activates the transcription program in host immune cells such as T cells via the intracellular signaling domain.

[0060] The intracellular signaling domain may include a domain involved in immune costimulatory signaling (e.g., a B7-binding domain) and, additionally or alternatively, an ITAM domain of CD3e. Preferably, the ITAM domain contains the amino acid sequence YMNM (SEQ ID NO: 4).

[0061] In some embodiments, both the transmembrane domain and the intracellular signaling domain are domains of the CD28 protein.

[0062] In some embodiments, the signaling domain includes an immunocostimulatory domain linked to a CD3e domain such as CD28 / CD3e, 4-1BB / CD3e, ICOS / CD3e, CD27 / CD3e, OX40 / CD3e, or CD40L / CD3e.

[0063] The labeled domain polypeptide is preferably a polypeptide that is not expressed or is minimally expressed in adult human tissue. For example, the labeled polypeptide may be derived from a protein (i.e., a "fetoprotein") that is expressed only in embryonic human cells or primarily in embryonic human cells, or the labeled polypeptide may be a completely synthesized amino acid sequence.

[0064] Examples of fetoproteins from which labeled polypeptides may be derived include fetoproteins expressed during embryonic development, such as Oct-4, Sox-2, and Klf-2. In some embodiments, proteins less than the full length are used, typically polypeptides of 20–100 aa in length. The amino acid sequences of Oct-4, Sox-2, and Klf-2 are as follows: Oct4: MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGI(Sequence ID 5) Sox-2: MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSR (Sequence ID 6) Klf-2: MALSEPILPSFSTFASPCRERGLQERWPRAEPESGGTDDDLNSVLDFILSMGLD (Sequence ID 7)

[0065] The labeling domain portion of ICAP may be a polypeptide having the amino acid sequence MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSF (SEQ ID NO: 8).

[0066] The ICAP-labeled domain may contain a structurally inactive domain of human mesoserine ECD. In the case of polypeptides encoding a mesoserine domain, it may contain the peptide sequence of domain I, II, or III below. EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL (Domain I - Sequence ID 9) SLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQN (Domain II - Sequence ID 10) CSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKL (Domain III - Sequence ID 11)

[0067] Labeled polypeptides can provide a "structurally inactive" domain of a structural membrane protein, provided that the labeled polypeptide originates from a structural membrane protein that does not have intracellular signaling function or interaction with other bioactive molecules and is usually bound by another protein or carbohydrate, so that the epitopes constituting the labeled domain are not exposed to antibodies in vivo. Labeled polypeptides preferably have little to no immunogenicity. The immunogenicity of labeled polypeptides can be determined by 1) an in silico computation algorithm for the number of T cell epitopes, 2) an in vitro assay to determine T cell activation ability, and 3) in vivo experiments using animal models.

[0068] Any of the above-mentioned labeling domains can be combined with any of the above-mentioned transmembrane domains and any of the above-mentioned intracellular signaling domains to form the ICAP polypeptide. The domains of ICAP can be linked using short polypeptide linkers.

[0069] For example, all of the above labeling domains can be encoded as the "labeling domain" portion of the plasmid pNB338B-ICAP-VHH shown in Figure 3B.

[0070] This construct is expressed in effector cells and produces an "immune cell activating polypeptide" (ICAP), which localizes to the extracellular membrane so that the labeling domain is extracellular.

[0071] The effector cells disclosed herein can be used with bispecific polypeptides, i.e., polypeptides having two functional domains linked by a linked polypeptide or by chemical conjugation, each domain having activity to specifically bind to a different ligand. In some embodiments herein, the bispecific polypeptide is also referred to as "VHH-TCP" as a preferred form of bispecific polypeptide comprising two or more single-chain nanobodies (single-chain, single-domain antibodies).

[0072] One domain of the bispecific polypeptide contains an amino acid sequence that specifically binds to the labeling domain (L-bd) of ICAP on the surface of effector cells, and the other domain of the bispecific polypeptide specifically binds to a protein on the surface of the “target,” which may be any cell or surface (CSP-bd) that is bound to the target protein, preferably a cellular target such as tumor cells. Such a surface-presented target polypeptide is referred to herein as the “cell surface protein” or its epitope.

[0073] Such cell surface proteins may be antigens associated with tumors, autoimmune diseases, or aging of cells or organisms, such as CD19, mesoserine, BCMA, EGFR, vimentin, Dcr2, or DPP4. In some embodiments, the target cells are cells that abnormally express one or more of these proteins in terms of quantity or as mutant proteins, such as B cells, mesothelial cells, mammary cells, or fibroblast tumors.

[0074] The bispecific polypeptides (VHH-TCP) used herein may include additional domains to provide additional binding activity, or biochemical or physiological activity, for example, to recognize multiple epitopes from the same target protein or epitopes from multiple target proteins (including "multispecific polypeptides" such as triplicate, tetraspecific, quinticate, or heptaspecific polypeptides). Bispecific polypeptides may also include one or more binding motifs for recognizing human IgGFc domains as labeling domains for ICAP that affect effector cell activity switchers via ADCC, CDC, and ADCP mechanisms.

[0075] Additionally or alternatively, bispecific (multispecific) polypeptides (VHH-TCPs) may also contain one or more domains derived from serum albumin of varying molecular weights to control the half-life of the bispecific polypeptide in vivo.

[0076] By including a domain that binds to a fluorescent dye in a bispecific polypeptide, it becomes possible to track the bispecific polypeptide and the cells to which it specifically binds in vivo (for example, by examining fluorescently stained tissue samples).

[0077] Preferably, the domains of the bispecific polypeptide can be linked to each other from the N-terminus to the C-terminus by one or more linker peptides. The length of the linkers can be adjusted to modulate (for example, to reduce) the molecular weight of the bispecific polypeptide or the steric interactions between its domains.

[0078] The linker portion of a bispecific polypeptide may also contain an amino acid sequence that is susceptible to cleavage by peptidases in the blood, thereby limiting the half-life of the bispecific polypeptide in the blood or extracellular matrix. For example, the amino acid sequences RVLAEA (SEQ ID NO: 12), EDVVCCSMSY (SEQ ID NO: 13), and GGIEGRGS (SEQ ID NO: 14) are cleavable by matrix metalloproteinase-1, and the amino acid sequence VSQTSKLTRAETVFPDV (SEQ ID NO: 15) is cleavable by factor IXa / factor VIIa.

[0079] In some embodiments, one or more, for example, all, of the active domains are composed of VHH nanobody polypeptides.

[0080] L-bd may be a single antibody domain derived from the VHH domain of camelid IgG. The CDR3 region of such a VHH domain may contain 15-20 amino acids that function as a paratope that binds to an epitope(s) on the label domain.

[0081] A bispecific polypeptide may include an L-bd, which is a VHH domain that specifically binds to a labeled polypeptide, and a CSP-bd, which is a VHH domain that specifically binds to CD19 or CD20. Such a bispecific polypeptide may be useful in treating B-cell lymphomas, such as non-Hodgkin lymphoma. In some embodiments, the bispecific polypeptide may include an L-bd, which is a VHH domain that specifically binds to a labeled polypeptide, and a CSP-bd, which is a VHH domain that specifically binds to EGFR. The amino acid sequence derived from the CDR3 region of the VHH antibody can bind to EGFR on the surface of non-small cell lung cancer cells. Such a bispecific polypeptide may be useful in treating non-small cell lung cancer.

[0082] A bispecific polypeptide may include an L-bd, which is a VHH domain that specifically binds to a labeled polypeptide, and a CSP-bd, which is a VHH domain that specifically binds to CPC3. In some embodiments, a bispecific polypeptide may include an L-bd, which is a VHH domain that specifically binds to a labeled polypeptide, and a CSP-bd, which is a VHH domain that specifically binds to BCMA. In some embodiments, a bispecific polypeptide may include an L-bd, which is a VHH domain that specifically binds to a labeled polypeptide, and a CSP-bd, which is a VHH domain that specifically binds to HER2. Such a bispecific polypeptide may be used with HER2 + It is useful in the treatment of breast cancer tumors.

[0083] An exemplary bispecific polypeptide comprising two linker-linked VHH domains (VHH bound to a label domain containing a structurally inactive peptide derived from human mesoserine ECD + linker + anti-EGFR VHH) has the amino acid sequence QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSS (SEQ ID NO: 16).

[0084] Preferably, the binding of the bispecific polypeptide to an epitope on cells other than the target cell does not have a significant effect on the pharmacokinetics or drug distribution of the bispecific polypeptide in vivo, and preferably, the resulting binding does not cause any significant observable physiological effect other than activating effector cells expressing the relevant labeling domain to which the bispecific polypeptide is bound.

[0085] By the embodiments illustrated and described herein, the applicant has devised methods and variations thereof for treating tumors using the engineered effector cells and bispecific polypeptides disclosed herein.

[0086] In one such method, engineered immune cells that can be T cells, or other cell types described herein as effector cells, are directly injected into solid tumors. Alternatively, the engineered immune cells can be administered intravenously (IV, for example, when treating leukemia or lymphoma). Depending on the disease indication, different administration methods may be implemented. In most cases, IV administration is carried out to treat the disease. Intraperitoneal administration can be carried out to treat malignant pleural mesothelioma (MPM).

[0087] In the treatment of solid tumors, direct injection into the tumor is expected to result in a better distribution of the cells within the tumor microenvironment (a larger number of engineered immune cells being proximal to the target tumor cells).

[0088] In a typical treatment method, the amount of VHH-TCP administered can be in the range of 10 ng / ml to 100 ng / ml together with engineered immune cells at a concentration of, for example, 5×10 4 、1×10 5 、5×10 5 、or 1×10 6 engineered cells / ml.

[0089] In an example embodiment of a treatment method that does not utilize a VHH-TCP activating molecule, the engineered immune cells are T cells expressing ICAP, which has a VHH-labeled domain that specifically binds to CD19 on B cells, as well as transmembrane domains and intracellular signaling domains for common T cell receptors (i.e., CD28 and CD3e). The engineered T cells also contain a vector for the expression of an anti-PD-1-Fc effector polypeptide under the control of a constitutive promoter. After administration of the cells to the target, the labeled-VHH domain of ICAP specifically binds to CD19 on B cells, and this binding signals the engineered immune cells, which are activated during intracellular signaling of CD3 and CD28 and proliferate near the B cell target. The proliferating cells secrete large amounts of anti-PD1 effector protein near the bound B cells.

[0090] The disclosed system offers one or more of the following advantages in various embodiments. Not all embodiments necessarily exhibit all of the advantages listed below.

[0091] In embodiments of the ICAP-labeled domain, polypeptides derived from fetoproteins or structural membrane proteins provide a wide range of L-bd for VHH-TCP binding. The lack of or low immunogenicity of the domain may improve the safety of cell therapies.

[0092] The diversity of domains that can be included in bispecific polypeptides (VHH-TCP) provides the ability to modify many properties, such as VHH-TCP affinity to effector cells, resulting in a broad range of cells that CSP-bd can target. Furthermore, other functional domains can be added, allowing for adjustment of epitope binding titers to suit the efficacy and safety of the system's use in treating diseases.

[0093] A bispecific polypeptide (VHH-TCP) that specifically binds to the labeling domain of an immune cell activator polypeptide containing a signaling domain that activates the proliferation of immune cell hosts, and specifically binds to B cell surface proteins such as CD19 ligands, can induce the proliferation of effector cells in vivo, increasing the population of immune effector cells and thus increasing the amount of effector polypeptide near B cell binding. This allows for reduced time and cost savings in the in vitro production of effector polypeptides.

[0094] Bispecific polypeptides (VHH-TCPs) can be manipulated in various forms to optimize effector cell activity through the length and flexibility of the linkers between VHHs in the bispecific polypeptide (VHH-TCP), the position of each binding motif, and the overall size of the VHH-TCM.

[0095] In vivo effector cell activity can be controlled by administering different amounts of bispecific polypeptide (VHH-TCP) and / or by controlling the half-life of VHH-TCP. This represents a novel, comprehensive approach to minimizing the toxicity of CAR-based therapies.

[0096] Furthermore, effector cells can be activated by the ADCC effect using appropriate labeled proteins that specifically bind to Fc epitopes.

[0097] Importantly, the characteristics of nanobodies, such as their small size, high stability, and ease of handling, offer unique advantages for optimizing in vivo treatment systems.

[0098] By discontinuing VHH-TCP administration to the target patient, adverse effects associated with sustained effector cell activity can be prevented, and if the disease recurs, an opportunity for subsequent VHH-TCP administration can be provided.

[0099] In situ secretion of antibodies, preferably nanobodies, by activated effector cells can inhibit or stimulate immune checkpoint receptors, thereby improving the targeting of solid tumors through penetration, proliferation, and persistence of the tumor microenvironment (TME). Nanobody bispecific polypeptides have advantages over conventional antibodies in TME penetration due to their small size and superior stability.

[0100] The effector cell-bispecific polypeptide (VHH-TCP) system disclosed herein can mitigate many of the risks associated with current CAR-T therapies, for example, by targeting multiple tumor antigens with a single standardized immune receptor, and the activity of immune cells can be controlled and optimized using diverse VHH-TCP structures. Treatments utilizing the disclosed system are expected to exhibit less toxicity or side effects. Furthermore, the components of the system are easily and cost-effectively manufactured. The diversity of ligands and binding domains that can be incorporated into ICAP and bispecific polypeptides (VHH-TCP) allows for the use of a modular system to treat or study a variety of diseases, for example, by incorporating a FITC-binding domain into VHH-TCP, the fate of activated effector cells can be tracked in vivo.

[0101] Examples Example 1 - Typical Operational Embodiment 1. Generation of modified effector T cells by electroporation ICAP consists of a labeled polypeptide (mesoserine or fetoprotein of 27aa), a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain (CD28IC), and CD3ζ. 1182-Fc(EQ) consists of a VHH-1182 domain and an IgG4Fc domain.

[0102] The 1182-Fc(EQ) structural gene is cloned into the piggyBac transposon vector pS338B to obtain the plasmid pS338B-1182-Fc(EQ) (Figure 3A). The ICAP-VHH gene is amplified by PCR and cloned into the piggyBac transposon vector pNB338B to obtain the plasmid pNB338B-ICAP-VHH (Figure 3B). The ICAP-VHH gene is replaced with an empty multi-cloning (MCS) gene to generate a MOCK construct plasmid.

[0103] Human peripheral blood mononuclear cells (PBMCs) from healthy donors are purchased from AllCells (Shanghai, China). The PBMCs are cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) in a 5% CO2 humidified incubator at 37°C for 0.5 to 1 hour, then harvested and washed twice with Dulbecco's phosphate-buffered saline (PBS).

[0104] PBMCs are counted, and 6 μg of pNB338B-ICAP-VHH plasmid or an equivalent volume of MOCK plasmid is electroporated in an electroporator (Lonza, Switzerland) using the Amaxa® Human TCell Nucleofector® kit according to the manufacturer's instructions. Subsequently, T cells transfected with ICAP-VHH / 1182-Fc(EQ) plasmid or MOCK / 1182-Fc(EQ) plasmid are specifically stimulated for 4-5 days in a 6-well plate coated with anti-CD3 antibody / anti-CD28 antibody (5 μg / mL). Next, the transformed T cells are cultured for 10 days in AIM-V medium containing 2% FBS and 100 U / mL recombinant human interleukin-2 (IL-2) to generate a sufficient quantity of effector T cells.

[0105] 2. Transduction Efficiency Assay The transduction efficiency of labeled polypeptides into T cells is determined by flow cytometry using biotin-conjugated anti-IgG4(Fc) antibody and PE-conjugated streptavidin secondary antibody.

[0106] 3. Binding efficiency assay The binding of bispecific polypeptides to typical T cells is measured by flow cytometry using an anti-CD19-PE antibody. Binding efficiency is determined by comparing the proportion of cells positive for CD19 and the label (e.g., meso).

[0107] 4. Proliferative capacity assay (culture with bispecific VHH and tumor cells) 1 x 10 7 Individual transformed T cells were pre-stained with carboxyfluorescein succinine midyl ester for 10 minutes, and then harvested in culture medium for another 10 minutes. (5 × 10 cells) 5 The cells are counted and co-cultured for 7 days with tumor cell lines expressing various antigens such as BCMA, EGFR, mesoserine, MUC1, and GPC3, as well as bispecific VHH, replacing the culture medium with fresh medium (AIM-V + 2% FBS) every 3-4 days. Next, the effector cells are assayed for proliferation by flow cytometry.

[0108] Quantification of 5.1182-Fc-VHH secretion 5 x 10 5 Individual cells were seeded in a 6-well plate containing 1 ml of culture medium, tumor cells and bispecific VHH were added, and co-cultured for 48 hours. Next, the effector T cell suspension was centrifuged at 3000 rpm for 3 minutes, the supernatant was retained, and the 1182-Fc protein was quantitatively detected by ELISA.

[0109] 6. Cytotoxicity assay (for adherent cell lines) The cytotoxicity of effector T cells transduced with a labeled construct or vector control is determined using an impedance-based xCELLigence RTCA TP Instrument.

[0110] Target tumor cells are seeded at a rate of 10,000 cells / well in a 96-well plate with a resistor at the bottom in an RTCA TP instrument for overnight (16 hours or more). Bispecific VHH antibody is added to the cultured target tumor cells, and they are cultured for a further 30 minutes. Next, transformed T cells containing plasmids pS338B-1182-Fc(EQ) and pNB338B-ICAP-VHH (effector cells) are incubated with target tumor cells for approximately 100 hours at different effector cell:target cell ratios (the endpoint depends on the killing efficiency of the transformed T cells). During the experiment, cell index values ​​closely correlate with tumor cell adhesion, indicating higher cytotoxicity with less cell adhesion, and are collected every 5 minutes by the RTCA system and an EnVision® multi-label plate reader (PerkinElmer). Real-time killing curves are automatically generated by the system software. The specific lysis (%) of each transformed T cell is also calculated using endpoint data [Specific lysis = (Cell index of tumor cells only - Cell index of transformed T cells co-cultured with tumor cells) / Cell index of tumor cells only].

[0111] 7. Cytotoxicity assay (for suspension cell lines) The cytotoxicity of effector T cells transduced with a labeled construct or vector control is determined according to the manufacturer's protocol (DELFIA® EuTDA Cytotoxic Reagent AD0116-PerkinElmer). Briefly, target tumor cells are washed with PBS and a fluorescence-enhancing ligand and incubated at 37°C for 5–30 minutes. 100 μl of target cells (10,000 cells) are placed in a V-bottom plate containing a bispecific polypeptide that specifically binds to both target tumor cells and effector cells (i.e., transformed T cells), and 100 μl of effector cells are added at various cell concentrations. After incubation at room temperature for 15 minutes, 20 μl of supernatant is transferred to 200 μL of europium solution. Fluorescence is measured using a time-resolved fluorometer. Specific release (%) = experimental release (number) - spontaneous release (number) / maximum release (number) - spontaneous release (number) × 100.

[0112] 8. Specific target activity in vivo NOD-SCID IL2 Rγ- / - (NSG) mice were generated under pathogen-free conditions (Shanghai, China). Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC). To establish a xenograft tumor model, 5 × 10⁶ NSG mice were implanted. 6 Individual EGFR + Lung tumor cells and 5 × 10 6 Individual MSLNs + Ovarian tumor cells are mixed with an equal volume of Matrigel (trademark) and subcutaneously inoculated. The tumor dimensions are obtained using calipers, and the tumor volume is calculated based on the following formula: V = 1 / 2 (length × width). 2 ) Tumor volume: approximately 100 mm 3 Therefore, Fluc-labeled effector T cells and bispecific EGFR-targeted VHH are injected intravenously. Specific targeting of effector T cells to the lungs is confirmed by bioluminescence imaging (BLI). On day 5, another bispecific MSLN-targeted VHH is injected intravenously to observe specific targeting of effector T cells to ovarian tumor cells. The proliferative capacity of effector T cells in vivo is monitored by bioluminescence imaging using the Xenogen IVIS imaging system (PerkinElmer, USA).

[0113] 9. In vivo proliferation and antitumor activity To establish a xenograft tumor model, NSG mice were given 5 × 10⁶ mice mixed with an equal amount of Matrigel (trademark). 6 Individual Fluc-labeled tumor cells are subcutaneously inoculated. The tumor volume is approximately 100 mm. 3 Therefore, mice are randomly divided into three groups (5 mice / group), and each group is intravenously injected (iv) with a PBS vehicle containing MOCK-T cells, effector T cells, or polypeptide VHH, with this point designated as day 0.

[0114] Peripheral blood from all mice is collected from the tail vein, and the proliferation of effector T cells and the copy number of the ICAP gene are detected. Mice that reach a mortal state are euthanized, and bone marrow, blood, and spleen are collected. CD3 in the above tissues. + The percentage of T cells and memory T cell subsets in the spleen will be analyzed by flow cytometry. Mouse body weight will be measured using an electronic balance throughout the entire in vivo experiment. Tumor progression will be confirmed by bioluminescence imaging (BLI) using the XenogenIVIS imaging system (PerkinElmer, USA). All measurements will be performed every 5 days.

[0115] 10. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) To evaluate the safety of cell therapy, H&E and immunohistochemistry will be performed. Mouse tissues (heart, liver, spleen, lung, kidney, and brain) will be fixed in formalin and embedded in paraffin. Using an RM2245 microtome (Leica, Germany), the tissues will be sequentially cut into 4 μm thick sections and stained with H&E. IHC analysis will be performed using a 1:100 dilution of anti-CD3 antibody (Abcam, #ab16669) to detect the invasiveness of effector T cells into tumor tissue. Images will be taken using an AXIOSTAR PLUS microscope (ZEISS, Germany).

[0116] 11. Tissue distribution assay The tissue distribution of 1182-VHH, transfected T cells, and adapter VHH protein is determined using quantitative real-time PCR (RT-qPCR). Mouse tissues (heart, liver, spleen, lung, kidney, and brain) are digested to prepare single-cell suspensions. Total DNA is extracted from T cells using the Genome DNA Extraction Kit Ver. 5.0 (TAKARA, China) according to the manufacturer's instructions. Real-time polymerase chain reaction is performed using TaqMan® Universal Master Mix II (ThermoFisher Scientific, USA). CAR and actin primers and probes are synthesized or labeled by Shanghai GenerayBiotech Co.Ltd (Shanghai, China). The quantitative real-time PCR reaction is performed in two steps: (1) pre-incubation: 5 minutes at 95°C; (2) amplification: 40 cycles of 20 seconds at 95°C, followed by 1 minute at 60°C. All reactions are performed in triplicate.

[0117] 12.Statistical analysis All data are expressed as mean ± SD. A t-test is used to assess the difference between two independent groups. One-way ANOVA is used to compare whether any statistically significant differences exist between three or more independent groups. Two-way ANOVA is used to determine the effect of two nominal predictors on a continuous outcome variable. All statistical analyses are performed using Graphpad Prism 7 version software (La Jolla, CA). All data, including error bars, are expressed as mean ± SD. Statistical significance is considered as follows: P ≥ 0.05 (ns), P < 0.05 (ns) * ), P<0.01( ** ), P<0.001( *** ), P<0.0001( **** ).

[0118] Example 2. Identification and characterization of a single VHH sequence with high affinity for MSLN, BCMA, or EGFR. We describe the identification and characterization of a single, specific VHH nanobody against MSLN, BCMA, or EGFR with high affinity using an alpaca immunotherapy library.

[0119] 1. VHH nanobodies relative to MSLNs In the initial immunization, each alpaca was subcutaneously administered 400 μg of MSLN-hFc emulsified with complete Freund's adjuvant. Two weeks later, 200 μg of MSLN-hFc emulsified with incomplete Freund's adjuvant was subcutaneously administered. Subsequently, five further immunizations were performed every other week with 200 μg of MSLN-hFc emulsified with incomplete Freund's adjuvant. High serum titers for both MSLN-His antigen and HEK293T-MSLN stable cell lines were confirmed by ELISA and FACS.

[0120] Seven days after the last injection, 50 mL of blood was collected, lymphocytes were purified from the sample, RNA was extracted and used to construct an immunotherapy library. Two rounds of solid-phase protein panning were performed using the MSLN-His antigen, followed by ELISA screening and FACS validation. One positive clone, named M2339(VHH), was obtained.

[0121] The antibody was expressed as an hFc fusion protein named M2339(VHH) using the procedure described in Patent Publication WO2020176815(A2) (which is incorporated herein by reference in its entirety for all purposes). The binding affinity of M2339(VHH) to the MSLN antigen was tested by surface plasmon resonance (SPR). First, M2339(VHH) was passed through a sensor chip pre-immobilized with protein A, and the antibody was captured by protein A. Next, five concentrations of MSLN-His protein were used as the mobile phase, with binding and dissociation times of 30 minutes and 60 minutes, respectively. On-rate (kon), off-rate (koff), and equilibrium constant (KD) were analyzed using Biacore evaluation software 2.0(GE). As shown in Table 1 below, the affinity of M2339(VHH) to the MSLN antigen was high, and K D The value was 2.64E-10.

[0122] The binding affinity of M2339(VHH) to HEK293T-MSLN cells was identified by flow cytometry. One 96-well plate was used to collect 3 × 10⁶ cells in each well, separating HEK293T cells from HEK293T-MSLN cells. 5 Cells were incubated in individual wells, followed by incubation with serially diluted M2339(VHH) for 30 minutes. Subsequently, the detection secondary antibody anti-human IgG PE (Jackson Immuno Research, code: 109-117-008) was incubated and detected using a CytoFLEX flow cytometer. "Isotype" is the isotype control (negative control). As shown in Figure 4A, M2339(VHH) showed excellent specific binding affinity to the HEK293T-MSLN cell line.

[0123] 2. VHH nanobodies against BCMA This example describes the identification and characterization of a single specific VHH nanobody for BCMA with high affinity using an alpaca immunotherapy library. The procedures for alpaca immunization, blood collection, library construction, solid-phase panning, ELISA or FACS screening of positive clones, antibody purification, and antibody characterization by SPR and FACS are described in Example 2 above. One positive clone named B029(VHH) was obtained.

[0124] As shown in Table 1, B029(VHH) has high affinity for the BCMA-His antigen, K D The value was 1.25E-10.

[0125] As shown in Figure 4B, B029(VHH) exhibited good and specific binding affinity to the CHOK1-BCMA cell line.

[0126] 3. VHH nanobodies against EGFR This example describes the identification and characterization of a single specific VHH nanobody for EGFR with high affinity using an alpaca immunotherapy library. The procedures for alpaca immunization, blood collection, library construction, solid-phase panning, ELISA or FACS screening of positive clones, antibody purification, and antibody characterization by SPR and FACS are described in Example 2 above. One positive clone named E454(VHH) was obtained.

[0127] As shown in Table 1, E454(VHH) has high affinity for EGFR His antigen, and K D The value was 1.27E-09.

[0128] As shown in Figure 4C, E454(VHH) exhibited good and specific binding affinity to the HEK293T-EGFR cell line. [Table 1]

[0129] Example 3. High-affinity VHH specific to regions II+III of mesoserine. In this embodiment, the identification and characterization of the label used in the present invention is described, which was identified as M2339(VHH) having similar affinity binding to mesoserine.

[0130] Different mesoserine ECD domains, including human Fc, were expressed in 293T cells and purified using a Protein A column. Affinity was determined by SPR. Various antigens were captured using a Protein A tip, and various concentrations of M2339(VHH) were injected at a flow rate of 10 μl / min. Binding times ranged from 120 to 180 seconds, and dissociation times ranged from 180 to 1200 seconds. Binding kinetics were determined using a 1:1 fit model with Biacore Evaluation software.

[0131] As shown in Figure 5, M2339(VHH) bound to complete mesoserine, mesoserine I, and mesoserine II+III with different affinities. The mesoserine II+III domain was well recognized by M2339, and its KD value was 4.32E-11M, showing similar affinity to the complete mesoserine polypeptide (Table 2). [Table 2]

[0132] These results demonstrate that M2339 binds to mesoserine II+III with high affinity and can be used as an adapter VHH, allowing mesoserine II+III to be used in ICAPs of T cells.

[0133] Example 4. Generation and screening of immune cell-activating polypeptides based on mesoserine II+III (M-ICAP). Various vectors, as shown in Figure 6, were constructed to generate so-called M-ICAP-T for activating immune cells such as T cells. All vectors encode the same intracellular region, including the 4-1BB and CD3ζ intracellular regions. The extracellular regions of the encoded polypeptides differed. M-ICAP does not contain any His-tag. M-ICAP-his-1 or 2 each contain a 6×His tag at either the N-terminus or C-terminus of M-ICAP, respectively. In addition to the mesoserine signal peptide (SP-MSLN), two other signal peptides (SPs) were selected from a human protein database to optimize expression rates. SP3-M-ICAP and SP5-M-ICAP contain different signal peptides, SP3 (MKHLWFFLLLVAAPRWVLS-SEQ ID NO: 1) or SP5 (MTRLTVLALLAGLLASSRA-SEQ ID NO: 2).

[0134] All vectors were transfected into 293T cells using Lipofectamin2000 (ThermoFisher, USA), and their expression rates were tested using flow cytometry after 2–4 days. For flow cytometry detection, blank cell controls were prepared using 19R73-CD19CAR and GFP vectors, with M2339-hFc and biotin-conjugated anti-His mAb used as primary antibodies, and fluorescent dye-conjugated anti-human Fc and fluorescent dye-conjugated streptavidin used as secondary antibodies. As shown in Table 3 and Figure 7, the position of the His tag affected the expression rate of the label (M-ICAP), with M-ICAP expression being higher when the N-terminal His tag was detected by M2339-hFc. The signal peptide had little effect on M-ICAP expression tested by both M2339-hFc and anti-His. [Table 3]

[0135] Example 5. Construction of M-ICAP-T cells As shown in Figure 8A, M-ICAP expression vectors containing different signal peptides (SP-MSLN, SP3, SP5) were constructed and fused with the T cell activation / signaling domains (CD28 / 4-1BB, CD3ζ) of a conventional CAR vector. The ICAP vector contained a labeled polypeptide M-ICAP (derived from the mesoserine II+III domain), a CD28 transmembrane domain, a CD28 / 4-1BB intracellular costimulatory signaling domain (CD28 / 4-1BBIC), and a CD3ζ domain. The ICAP-VHH gene was amplified by PCR and cloned into the piggyBac transposon vector pNB338B to obtain the plasmid pNB338B-ICAP-VHH (Figure 8B).

[0136] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased from AllCells (Shanghai, China). The PBMCs were cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) in a 5% CO2 humidified incubator at 37°C for 0.5–1 hour, then harvested and washed twice with Dulbecco's phosphate-buffered saline (PBS). The PBMCs were counted, and 6 μg of M-ICAP, SP3-M-ICAP, and SP5-M-ICAP vectors were electroporated using an Amaxa® Human TCellNucleofector® kit according to the manufacturer's instructions in an electroporator (Lonza, Switzerland). Subsequently, transfected T cells were specifically stimulated for 4-5 days in 6-well plates coated with anti-His / M2339 (VHH-Fc) and anti-CD28 antibodies (5 μg / mL), and cultured for 10 days in AIM-V medium containing 2% FBS and 100 U / mL recombinant human interleukin-2 (IL-2) to generate a sufficient amount of effector T cells. Transduction efficiency of labeled polypeptides on T cells (M-ICAP expression) was determined by flow cytometry using biotin-conjugated anti-His antibody and PE-conjugated streptavidin secondary antibody.

[0137] As shown in Figures 8C and 8D, after amplification, the positivity rate of M-ICAP-T cells all exceeded 30% on day 8 and 92% on day 13. All three different ICAPs were activated and amplified by stimulation with M2339VHH or anti-His antibody. The expression of M-ICAPECD outside the T cell membrane and the construction of M-ICAP-T cells were successful.

[0138] Example 6. Generation and validation of M-ICAP-T cells M-ICAP was fused to several different CAR sequences, and the resulting M-ICAP-T cells were obtained by electroporation combined with specific activation using donor-derived PBMC cells. The ICAP vector contained a labeled polypeptide (derived from the mesoserine II+III domain), a CD28 transmembrane domain, a CD28 / 4-1BB intracellular costimulatory signaling domain (CD28 / 4-1BBIC), and a CD3ζ domain. 1182-Fc(EQ) contained the VHH-1182 domain and the IgG4Fc domain.

[0139] The generation of ICAP CAR-expressing cells (ICAP-T cells) or classical CAR-T cells by electroporation is described in Example 5.

[0140] After amplification, a series of tests were performed to validate the modified T cells, including the positivity of ICAP expression, amplification effect, the ratio of CD4 / CD8-positive cells in CD3-positive cells, and the ratio of effector memory T (Tem) cells to central memory T (Tcm) cells in memory T (Tm) cells. The expression rate of labeled polypeptide on the T cell surface (M-ICAP expression) was determined by flow cytometry using biotin-conjugated anti-His antibody and PE-conjugated streptavidin secondary antibody. As shown in Figure 9, amplification of ICAP-T cells being prepared from PBMCs of two donors (AC1909A and SL2007A) was up to 10-fold, ICAP expression rates were up to 80% (depending on the donor source), CD4 / CD8 positivity varied depending on the different donors, and central memory T cells constituted the majority of memory cells. Different CAR element sequences had some effect on the positivity and amplification of ICAP-T cells. For example, compared to M-ICAP and M-ICAP-28, M-ICAP-28BB-T cells showed less proliferation and ICAP expression. Regarding the specific activation of T cells, the inventors compared the effects of various antibodies / TCPs on the amplification of M-ICAP-transfected PBMCs. As shown in the figure, M2339, anti-His antibody, and TCP001-C / P were able to specifically activate the growth of ICAP-T cells.

[0141] Example 7. TCP design and characterization based on M2339VHH This embodiment describes the design and characterization of the TCP used in this application. The TCP used herein is a bispecific antibody capable of simultaneously recognizing target B cells or tumor-specific antigens (such as CD19, BCMA, and EGFR) and the M-ICAP polypeptide (deserine-derived) of M-ICAP-T cells, and can therefore be used as an adapter to control the proliferation or cytotoxicity of M-ICAP-T cells. The TCP designed and applied in the current working embodiment is shown in Table 4. [Table 4]

[0142] 1. TCP design and refinement For use in cytotoxicity assays and in vitro efficacy assays, as further described below, we designed BCMA-TCPs capable of simultaneously targeting BCMA antigen and M-ICAP polypeptide (labeled with mesoserine). To investigate the effects of various linker formats on the bioactivity and stability of TCPs, we designed three formats of BCMA-TCP (TCP001-C, TCP002-C, and TCP003-C) with different linkers (3×GGGGS linker, hIgG4-Fc, and hIgG4-CH3, respectively). TCP001-P and TCP001-N served as positive and negative controls for the TCP format, respectively. Simultaneously, MC001C and MC001D, targeting BCMA and M-ICAP respectively, were constructed as two positive controls in the mAb format.

[0143] We designed a single CD19-TCP, named TCP011-P, which simultaneously targets the CD19 antigen and M-ICAP polypeptide via a 3×G4S linker, for use in a CD19 antigen-stimulated M-ICAP-T proliferation assay.

[0144] We designed a single EGFR-TCP, named TCP021-P, which simultaneously targets the EGFR antigen and M-ICAP polypeptide via a 3×G4S linker, for use in an M-ICAP-T cytotoxic assay targeting EGFR-expressing solid tumor cell lines.

[0145] The N-terminal M2339VHH sequence targeting the M-ICAP polypeptide was identified from phage display using the alpaca immunoVHH library described in Examples 2 and 3 above. The B029(VHH) sequence targeting BMCA was identified from phage display using the alpaca immunoVHH library described in Example 2 above. The scFv sequence within TCP001-P targeting BMCA was derived from B2121 of CN 201580050638. The VHH sequence of TCP001-N targeting GFP was derived from GFP-specific VHH described by Kubala et al. (MH Kubala et al, Protein Sci. 19:2389-2401 (2010) (a description of these VHHs and their uses is incorporated in their entirety by reference)).

[0146] The scFv sequence within TCP011-P, which targets CD19, was derived from FMC063, as described in Chinese Patent Application CN No. 201480027401.4 (the whole sequence is incorporated by reference for all purposes). The E454 sequence within TCP021-P, which targets EGFR, was identified from phage display using the alpaca immunoVHH library described in Example 2 above.

[0147] These genes were synthesized and cloned by Genewiz, Inc. All ORF DNA was cloned between the BamHI and EcoRI sites into the pcDNA3.4 vector. Antibody expression, purification, and purity quality control were carried out as described in publication WO2020176815A2 (a description of such methods is incorporated herein by reference).

[0148] 2. Affinity Characterization of TCP First, the binding affinity of purified BCMA-TCP to BMCA antigen was evaluated by SPR. BCMA-his antigen was bound to a CM5 chip (GE Healthcare Life Sciences), and various anti-BCMA BsAbs were flowed at a flow rate of 10 μL / min with a dissociation time of 900 seconds. Binding kinetics were determined using a 1:1 fit model. The data showed that TCP001-C, which has a 3×G4S linker, had a higher binding affinity than TCP002-C and TCP0031-C, which have larger linkers, suggesting that linker structure may influence binding affinity (Table 5). [Table 5]

[0149] The binding bioactivity of mesoserine and BCMA-overexpressing cells was evaluated by flow cytometry. Stable cell lines were recovered using 0.25% trypsin.

[0150] Approximately 5E5 cells were collected per sample, and the cells were resuspended in 100 μL / well of His-tagged test antibody. The cells were then incubated with anti-His-tagged antibody (Genscript, China) and streptavidin-PE (Biolegend, China). Each incubation step was performed in the dark at 4°C for 1 hour, after which the cells were washed twice with 200 μL of PBS buffer. The washed cells were resuspended in 200 μL of PBS buffer, and the samples were analyzed by FACS. As shown in Figure 10, TCP002-C bound most strongly to both cell lines, although the binding strength of TCP003-C was slightly higher than that of TCP001-C.

[0151] 3. Stability of BCMA-TCP in human plasma in vitro TCP001C, TCP002C, and TCP003C were incubated in 100% human plasma at 37°C for up to 21 days, with samples collected on days 0, 1, 3, 7, 14, and 21, respectively. 96-well plates were coated with mesoserine antigen, blocked, and washed. The collected samples, appropriately diluted, and the serially diluted standard samples were incubated with the plates at 37°C for 1 hour. Anti-VHH-cocktail-HRP (GenScript, A02016) was used as the detection antibody, and absorbance was read at 450 nm. Finally, the test samples were analyzed according to a curve fitted to the standard sample group.

[0152] As shown in Figure 11, TCP001-C, TCP002-C, and TCP003-C are stable in human plasma for longer than 21 days at 37°C in vitro.

[0153] The binding affinity of TCP011-P to cells overexpressing both CD19 and MSLN (Figure 12), and the binding affinity of TCP021-P to cells overexpressing both EGFR and MSLN (Figure 13), were confirmed by flow cytometry using the same procedure applied to BCMA-TCP.

[0154] Example 8. In vitro amplification of M-ICAP-T by TCP directed at target cells To verify the rapid activation and amplification of ICAP-T cells by TCP culture with target cells, PBMC-T cells transfected with M-ICAP (activated by anti-His and anti-CD28) were co-cultured with CD19-positive Daudi lymphoma cells in the presence of TCP011-P or TCP011-N, respectively. CD19-positive Daudi lymphoma cells were treated for 2 hours in or without 50 μg / ml mitomycin C. 5 × 10⁶ M-ICAP cells were then transfected. 5 We counted individual PBMC-T cells, 5 × 10⁶ 5Daudi cells were co-cultured with TCP011-P or TCP011-N for 4 days. The effector or target cells were then analyzed for proliferation by flow cytometry.

[0155] As shown in Figure 14, M-ICAP-T cells transfected for 5, 8, and 13 days in the presence of TCP011-P effectively increased in size when stimulated by Daudi cells, with maximum amplification occurring on days 5 and 8 post-transfection. Furthermore, activated M-ICAP-T cells were able to kill Daudi cells.

[0156] Example 9. TCP dose-dependent cytotoxic effect of M-ICAP-T on RPMI-8226 cells. For ICAP-T cells to act on BCMA-positive tumor cells, they need to have a TCP that can specifically bind to BCMA. The two ends of TCP001-C and TCP001-P can simultaneously specifically bind to both ICAP-T cells and BCMA cells. To confirm that ICAP-T cells can act on BCMA-positive tumor cells and have a specific cytolytic / killing effect in combination with a specific TCP, we compared the cytolytic / killing effects of ICAP-T cells or CAR-T cells (at three different E:T ratios) co-cultured with RPMI-8226 cells or L363 cells in the presence of different TCPs.

[0157] T cell cytotoxicity assays of suspension cell lines were performed according to the manufacturer's protocol (DELFIA® EuTDA cytotoxic reagent AD0116-PerkinElmer). Briefly, target tumor cells were washed with PBS and a fluorescence-enhancing ligand and incubated at 37°C for 15 minutes. 50 μl of target cells (5,000 cells) were placed in a V-bottom plate containing a bispecific polypeptide that specifically binds to both target tumor cells and effector cells (i.e., transformed T cells), and 50 μl of effector cells were added at various cell concentrations (E:T = 16 / 8 / 4:1). After 3.5 hours of incubation, 10 μl of supernatant was transferred to 100 μL of Europium Solution. After incubation at room temperature for 15 minutes, fluorescence was measured using a time-resolved fluorometer. Specific release (%) = experimental release (number) - spontaneous release (number) / maximum release (number) - spontaneous release (number) × 100.

[0158] T cell secretion of IFNγ was also assayed. IFNγ detection was performed according to the manufacturer's protocol (IFNγ detection kit, VAL104-Novus). Briefly, fresh washing solution, colorant, diluent, and standard products were prepared according to the instructions. Standard solutions of different concentrations and diluted experimental samples were added to the corresponding wells at 100 μl / well. The reaction wells were sealed with sealing tape and incubated at room temperature for 2 hours. After four washes with washing buffer, 200 μL of enzyme-labeled antibody was added to each well and incubated at room temperature for 2 hours. After repeating the plate washing procedure, 200 μL of pre-mixed colorant was added to each well and the reaction was incubated in the dark for 10–30 minutes. By adding 50 μl / well of stop solution, the color of the solution changed from blue to yellow. OD values ​​were recorded with a spectrophotometer for 20 minutes and analyzed in Excel using the selected "four-parameter equation" to obtain a standard curve using the standard sample group.

[0159] As shown in Figure 16, M-ICAP-T combined with TCP001-C showed a strong specific killing effect against tumor target cells, but the nonspecific killing effect of T cells was more pronounced at E:T=16:1. When the TCP concentration exceeded 0.025 μg / ml, the cytolytic effects of E:T=8:1 and 4:1 increased. At TCP001-C concentrations of 0.1 and 0.5 μg / ml, E:T=16:1 and 8:1 showed the best killing effect. When the TCP001-C concentration reached 2 μg / ml, the effects of E:T=16:1 and 8:1 actually decreased. E:T=16:1 showed the best killing effect. For different E:T ratios, the TCP EC50 values ​​were similar (EC50 = 0.028 (E:T = 16:1), 0.024 (E:T = 8:1), 0.022 (E:T = 4:1)), but the maximum value corresponded to the E:T ratio.

[0160] Example 10. Comparison of cytotoxicity of ICAP-T combined with different TCPs and detection of IFNγ secretion in RPMI-8226 / L363 cells. The two ends of TCP001-C / P, TCP002-C / P, and TCP003-C / P simultaneously bind to M-ICAP and BCMA. To confirm and compare the specific cytolytic effects of ICAP-T cells combined with these TCPs against BCMA-positive tumor cells, cytolysis / killing assays of ICAP-T cells or CAR-T cells co-cultured with RPMI-8226 or L363 cells were performed in the presence of various TCPs (E:T=8:1). T cell cytotoxicity and IFNγ secretion assays against suspension cell lines are described in Example 9.

[0161] As shown in Figure 16, M-ICAP-T combined with TCP001-C exhibits a potent specific killing effect against tumor target cells, while combinations of TCP001-C (binding only to BCMA-positive cells) or TCP-MD (no binding to BCMA-positive cells) cannot effectively kill FaDu / SK-OV3 cells. Furthermore, IFNγ secretion data are consistent with cytotoxic data. Combining M-ICAP-T with both TCP001-C / P at 0.2 μg / ml specifically induced IFNγ release in RPMI-8226 and L363 cell lines compared to negative control groups (TCP001-N, TCP-MD, or IgG). The release order was TCP001 > TCP003 > TCP002.

[0162] Example 11. Cell lysis effect of ICAP-T cells combined with TCP (EGFR binding) against FaDu / SK-OV3 cells For ICAP-T cells to act on EGFR-positive tumor cells, they need to possess a TCP that can specifically bind to EGFR. TCP021-P can combine ICAP and EGFR at its two ends, respectively. To confirm that ICAP-T cells combined with this specific TCP can act on EGFR-positive tumor cells such as FaDu (human pharyngeal squamous cell carcinoma) and SK-OV3 (human ovarian cancer cells), the toxic effects of ICAP-T cells or CAR-T cells co-cultured with FaDu / SK-OV3 cells were compared in the presence of various TCPs.

[0163] T-cell cytotoxicity assays against adherent cell lines are performed using impedance-based RTCA TP instruments and methods (xCELLigence). Target tumor cells are seeded at 10,000 cells / well overnight (16 hours or more) in an RTCA TP instrument in a 96-well plate with a resistor at the bottom. Bispecific TCP or antibody is added to the cultured target tumor cells and cultured for a further 30 minutes. Next, ICAP-T cells or CAR-T cells are incubated with target tumor cells for approximately 100 hours at different effector cell:target cell ratios (the endpoint depends on the killing efficiency of the transformed T cells). During the experiment, cell index values ​​are closely related to tumor cell adhesion, indicating higher cytotoxicity with less cell adhesion, and are collected every 5–10 minutes by the RTCA system. Real-time killing curves are automatically generated by the system software. The specific lysis (%) of each transformed T cell is also calculated using data at 48 hours [Specific lysis = (Cell index of tumor cells only - Cell index of transformed T cells co-cultured with tumor cells) / Cell index of tumor cells only].

[0164] As shown in Figure 17, M-ICAP-T combined with TCP021-P exhibits a strong specific killing effect against two different tumor target cells, similar to EGFR CAR-T (the nonspecific killing effect of T cells is more evident at E:T=4:1), whereas combinations of TCP001-C (binding only to BCMA-positive cells) or TCP-MD (not binding to EGFR-positive cells) cannot effectively kill FaDu or SK-OV3 cells.

[0165] Example 12. IFN-γ release and cytolytic effect of TCP-containing ICAP-T cells on Daudi cells For ICAP-T cells to act on B cells, they need to possess a TCP that can specifically bind to CD19. TCP011-P can bind to ICAP and CD19 at its two ends, respectively. To confirm the effect of ICAP-T cells combined with this specific TCP on CD19-positive B cells, we compared the cytotoxicity and IFN-γ release of Daudi cells when ICAP-T cells or CAR-T cells were co-cultured with Daudi cells in the presence of various TCPs. T cell cytotoxicity and IFN-γ secretion assays for suspension cell lines are described in Example 9.

[0166] As shown in Figure 18, IFN-γ secretion showed a significant difference. M-ICAP-T combined with TCP011-P was similar to CD19CAR-T in terms of cell killing and IFN-γ secretion. TCP001-C (bound to BCMA) exhibited a cell-killing effect against Daudi cells, but the level of IFN-γ secreted by T cells was significantly reduced. Furthermore, when combined with TCP-MD, it was unable to effectively secrete IFN-γ (unable to bind to Daudi cells).

[0167] Example 13. Generation and characterization of VHH secreting ICAP-T. Due to the complex tumor microenvironment, most CAR-T therapies targeting solid tumors currently being tested clinically have shown little clinical efficacy. To enhance the antitumor effect of the ICAP-T cell lineage, M-ICAP-T cells that secrete immune checkpoint inhibitors, such as anti-PD-1 (an antagonist of tumor suppressor cytokines like anti-TGFβ), were produced by simultaneously transfecting human naive T cells with M-ICAP peptide (derived from MII+III peptide) and plasmids encoding secretory immune checkpoint inhibitors.

[0168] Thirteen days after M-ICAP-T preparation, FACS was used to detect ICAP expression. The results are shown in Figure 19. Compared to the M-ICAP-T control, the nature of the protein secreted as VHH or scFv did not have any clear effect on ICAP expression.

[0169] The concentrations of secreted proteins were tested using ELISA. The supernatant was added to a 96-well plate coated with the antigen, and HRP-bound anti-VHH and HRP-anti-His were used to detect anti-PD-1 VHH, anti-PD-L1 VHH, and anti-TGFβ scFv, respectively. The results are shown in Figure 19B, where the concentration of secreted VHH was approximately 75 ng / ml and the concentration of secreted anti-TGFβ scFv was approximately 150 ng / ml.

[0170] Example 14. Secretion of anti-PD-1VHH blocked the surface expression of PD-1. The binding ability of secreted anti-PD-1VHH was indirectly tested by FACS on T cells expressing PD-1 on their surface. PD-1 expression levels on T cells were examined in a competitive assay using commercially available anti-PD-1 antibodies. As shown in Figure 20, the supernatant of ICAP-T cells secreting anti-PD-1 blocked the binding of commercially available anti-PD-1 antibodies to human primary T cells stimulated by CD3 and CD28.

[0171] Example 15. Anti-TGFβscFv secreted by M-ICAP-T cells blocked TGFβ-1-induced luciferase cell signaling. The blocking activity of the secreted anti-TGFβscFv obtained in Example 13 was determined using a commercially available TGFβRII-293T-Luc cell line. 5000 TGFβRII-293 cells were seeded and incubated overnight. After adding the test sample, 5nMTGFβ was added, and bioluminescence was read using ONE-GLO after 6 hours. The results are shown in Figure 21. Anti-TGFβscFv secreted by M-ICAP T cells blocked the luciferase signaling induced by TGFβ. CAR-T-10C, 10B, and 01A are anti-TGFβM-ICAP-T cells prepared from different donors.

[0172] Example 16. In vivo efficacy study of M-ICAP-T used with TCP001-C in an orthotopic tumor model of L363-PDL1-LUC. In the orthotopic tumor model experiments described below, NPSG mice (NOD-Prkdc scid IL2rg tm1 Use / Pnk).

[0173] 1. Tumor inoculation, grouping, drug administration, and animal observation. (a) L363-PDL1-luc tumor cells were cultured at 37°C in an atmosphere of 5% CO2 air in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells growing in the exponential growth phase were collected and counted for tumor inoculation. The cell count at inoculation was 4.09E+8, and the viability was 83.65%. (b) For efficacy testing, each mouse was given 2 × 10 6200 μL PBS containing L363-PDL1-luc cells was IV-inoculated. The tumor inoculation day was defined as day 0. On day 8, when the tumor volume reached approximately 9.4E5, 24 mice were selected and randomly grouped into 7 groups according to the animals' body weight and tumor volume. Each group had 3-5 tumor-bearing mice. The 7 groups were established based on the category of administered drug, dosage, and frequency of administration. Group 1 was set up as a negative control group that received only PBS throughout the entire study phase. Group 2 was another negative control group, receiving a high dose (20 * E6) anti-PD-1 M-ICAP-T cells were intravenously injected on day 8, followed by seven subcutaneous injections of PBS every two days. Group 3 was another positive control group, and 5 * E6 classical BCMA CAR-T (B2121) was administered intravenously on day 8, followed by seven subcutaneous injections of PBS every two days. Groups 4 and 5 were two experimental groups, each receiving a low dose (5 * E6) and high dose (20 * E6) anti-PD-1 M-ICAP-T cells were injected, followed by 7 subcutaneous injections of 5 mg / kg of TCP001-C every two days. Groups 6 and 7 were two separate experimental groups, each consisting of 5 * E6 and 20 * E6 M-ICAP-T cells were injected, followed by seven subcutaneous injections of 5 mg / kg of TCP001-C every two days. (c) All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, accreditation number 001516), approved by the Institutional Animal Care and Use Committee (IACUC) of BioDuro. At the time of periodic monitoring, animals were checked for any adverse effects of tumor growth and / or treatment on normal behavior, including motility, food and water consumption (by observation only), and weight gain or loss (weight was measured twice a week in the pre-treatment phase and daily in the treatment phase), effects on eyes / hair tangles, and any other abnormal effects such as tumor ulcers. Any animal was notified to the study sponsor when weight loss reached 10%.

[0174] 2. Body weight; tumor measurement Body weight and bioluminescence signals were measured twice a week. The results of body weight changes in tumor-bearing mice are shown in Figure 22. No abnormal body weight changes were observed in any group during the experiment. The bioluminescence signal in mice was measured twice a week throughout the study, starting on day 4 after cell injection using IVIS Lumina XR. The signal was quantified using Living Image software. As shown in Figure 23, there was no significant difference in efficacy between group 2 (M-ICAP-T injected only after tumor inoculation) and group 1 (tumor inoculation only) during the study period (days 8-26). In comparison, all experimental groups (groups 4, 5, 6, and 7) injected with conventional TCP001-C-activated M-ICAP-T showed significant efficacy against L363-PDL1 in the orthotopic tumor model compared to the two negative control groups (groups 1 and 2). Furthermore, the experimental groups (groups 4, 5, 6, and 7) showed similar efficacy to classical BCMA CAR-T therapy (group 3).

[0175] In the established L363-PDL1-LUC orthotopic tumor model, tumor growth can be significantly suppressed by injecting M-ICAP-T cells every other day along with the usual TCP001-C injection.

[0176] 3. Analysis of anti-PD-1 and TCP001-C concentrations in mouse whole blood To analyze the concentrations of anti-PD-1 and TCP001-C in whole mouse blood, 100 μl of peripheral blood was collected weekly. For the ELISA binding assay, 1 μg / ml of PD-1 protein was coated overnight into 96-well plates. Diluted samples and diluted standard samples (8 dilution points from 2 ng / ml) were added to the wells and left at 37°C for 1 hour. Then, an anti-VHH cocktail antibody was added as the detection antibody, and the assay reagents were added. Absorbance was read at 450 nm. The concentrations were determined by analysis against a standard curve as in Example 9.

[0177] As shown in Figure 24, anti-PD-1 VHH was significantly detectable only in groups 2, 4, and 5 at two sampling time points (day 15 and day 22). Groups 2, 4, and 5 used anti-PD-1 M-ICAP-T cells as effector T cells, indicating that PD-1 can be successfully secreted in vivo by anti-PD-1 M-ICAP-T cells.

[0178] The method for analyzing TCP001-C concentration is described in the "Assessment of Antibody Stability in Human Plasma" section (Example 5) of the Antibody Production and Characterization part. High concentrations of TCP001-C can be detected in peripheral blood collected 24 and 48 hours after subcutaneous injection of TCP001-C. This indicates that the half-life of TCP001-C in vivo is longer than 48 hours, and that an injection frequency of once every two days is sufficient to support the efficacy of M-ICAP-T against L363 tumor cells.

[0179] Example 17. Identification and Characterization of Exemplary BCMA Peptide Motifs as a General-Purpose Tagging System for In vitro and In vivo Amplification of CAR-T A peptide motif (approximately 20–30 aa in length) was fused to the N-terminus of the antigen-binding domain (scFv or VHH) of the CAR-T cell receptor as a general-purpose ICAP for CAR-T amplification in vitro or in vivo. The following criteria were used for designing the peptide motif.

[0180] First, the peptide is approximately 20–30 aa in length. Next, a nanobody specific to this peptide motif with high binding affinity (KD < 1 nM) can be obtained. Finally, when the peptide-targeting nanobody fuses the peptide to the N-terminus of the antigen-binding domain (scFv or VHH) of the chimeric antigen receptor of CAR-T cells, it successfully induces CAR-T amplification in vitro or in vivo.

[0181] 1. Identification and characterization of a single VHH sequence with high affinity for MSLN. Using an alpaca immunotherapy library, one VHH nanobody with high affinity for MSLN was identified. The procedures for alpaca immunotherapy screening, blood collection, library construction, solid-phase panning, ELISA or FACS screening of positive clones, antibody purification, and antibody characterization by SPR and FACS were as described in Example 2. One positive clone, named anti-MSLN-1444VHH, was obtained.

[0182] As shown in Table 6, anti-MSLN-1444 VHH showed high affinity for the MSLN His antigen, with a KD of 2.10E-09. [Table 6]

[0183] As shown in Figure 25, anti-MSLN-1444 VHH exhibited excellent specific binding affinity to HEK293T-MSLN cells.

[0184] 2. Identification of BCMA peptides (BCMA ICAP) that can be strongly recognized by full-length BCMA VHH binders. We designed a BCMA peptide motif of an appropriate length (approximately 20 aa) that could be recognized with high affinity by several BCMA candidate binding proteins from a previously prepared immunolibrary containing the BCMA-hFc antigen, as well as by many candidate VHH sequences with varying binding properties to full-length BCMA. We selected one polypeptide of natural BCMA, BCMA mut1 (1-23 aa of the BCMA ECD domain, Table 7-SEQ ID NO: 17), and Figure 26 shows a fusion polypeptide of BCMA mut1 with an anti-MSLN-1444VHH sequence that targets expressed MSLN.

[0185] Next, the inventors excluded three VHH sequences (#36, #102, and #367, described below) that had high affinity for BCMA mut1. The sequence of BCMA mut1 is also shown in Table 7 (SEQ ID NO: 18). The three VHH sequences were represented as hFc fusion proteins named 36(VHH), 102(VHH), and 367(VHH) according to the procedure described in Japanese Patent Publication WO2020176815A2 (which is incorporated herein by reference in its entirety for all purposes). The binding affinity of the three VHH nanobodies was measured by SPR. As shown in Figure 27, the three VHHs showed high affinity binding to BCMA mut1. The binding kinetic parameters of these three VHHs are shown in Table 8. Anti-BCMA VHH 36# was selected as a stimulator due to its high affinity for BCMA ICAP BMCAmut1. [Table 7] [Table 8]

[0186] 3. BCMA ICAP can be used to specifically amplify CART cells. The ICAP-1-23-3GS vector was constructed as shown in Figure 28A, and had BCMAmut1(ICAP) at the N-terminus of an anti-MSLN CAR linked by a (G4S)3 linker. BCMAmut1-MSLN-1444 CAR-T cells were prepared by transfection with the BCMAmut1-MSLN-1444 vector and then stimulation with plate-coated MSLN and anti-CD28 or anti-BCMAmut1 36# and anti-CD28, respectively. After 9 days of culture, the CAR-T cells showed amplification activity comparable to that of anti-BCMAmut1 36#-stimulated cells in two donors, compared to CAR-T cells stimulated with the antigen MSLN (Figures 28B, 28C, and 29A, 29B).

[0187] 4. Anti-BCMAmut136# did not stimulate nonspecific amplification of CART cells by BCMA ICAP. To test the specificity of anti-BCMAmut136# stimulation of BCMA ICAP CAR-T cells, the MSLN-1444 CAR vector was constructed as shown in Figure 30A. After transfection of PBMCs, cells were prepared by stimulation with MSLN and anti-CD28 coated on plates, or with anti-BCMAmut136# and anti-CD28, respectively. The results are shown in Figure 30B. Only CAR-T cells stimulated with MSLN and anti-CD28 showed clear amplification. MSLN-1444 CAR cells stimulated with anti-BCMA36# and anti-CD28 did not show amplification in two donors.

[0188] Additional nucleic acid and amino acid sequences MSLN region II + region III (M3) DNA: TCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCAACAAAGGGCACGAAATGAGTCCTCAGGTGGCCACCCTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACACCCTGACCGCCTTCTACCCTGGGTACCTGTGCTCCCTCAGCCCCGAGGAGCTGAGCTCCGTGCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTGTGACCCAAGGCAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGTCCGAATACTTCGTGAAGATCCAGTCCTTCCTGGGTGGGGCCCCCACGGAGGATTTGAAGGCGCTCAGTCAGCAGAATGTGAGCATGGACTTGGCCACGTTCATGAAGCTGCGGACGGATGCGGTGCTGCCGTTGACTGTGGCTGAGGTGCAGAAACTTCTGGGACCCCACGTGGAGGGCCTGAAGGCGGAGGAGCGGCACCGCCCGGTGCGGGACTGGATCCTACGGCAGCGGCAGGACGACCTGGACACGCTGGGGCTGGGGCTACAGGGCGGCATCCCCAACGGCTACCTGGTCCTAGACCTCAGCATGCAAGAGGCCCTCTCG(SEQ ID NO:19) MSLN domain II + domain III (M3) protein: SLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALS(SEQ ID NO:20) M-ICAP CAR ORF DNA: M-ICAP CAR ORF protein: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPHHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLATAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILR QRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(sejq 22) M-ICAP-SP3 CAR ORF DNA: M-ICAP-SP3 CAR ORF Protein: MKHLWFFLLLVAAPRWVLSHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24) M-ICAP-SP5 CAR ORF DNA: M-ICAP-SP5 CAR ORF protein: MTRLTVLALLAGLLASSRAHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLD TCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLD TLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 26) M(2339VHH)DNA sequence: CAGCTGCAGCTGGGCGCCTCTGGCGGCGGCCTGGTCCAGCCTGGCGGCTCTCTGAGACTGAGCTGTGCCCTGTCTGGCTTCACACTGAGAGAGCTGGACGAGTTCGCCATCGGCTGGTTCAGGCAGGCCCCTGGCAAGGAGAGAGAGGGCGTGAGCTGTATCAGCGGCACAGGCGGCATCACACATTATGCTG ACAGCGTGAAGGGCAGGTTCACAATCAGCAGAGACATCGCCAAGACAACCGTGTACCTGCAGATGAATAGCCTGAACAGCGAAGACACAGCCGTGTACTACTGTGCCGCCGACGAGAGATGTACAGACAGACTGATCAGACCTCCTACATATTGGGGACAAGGCACCCAGGTGACAGTCTCTTCT (SEQ ID NO: 27) M(2339VHH) protein sequence: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSS (Sequence ID 28) BCMA B029(VHH) sequence in TCP001-C and MC001C: QVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSS (Sequence ID 29) CD19 scFv sequence in TCP011-P (CN201480027401.4.): DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(Sequence ID 30) EGFR E454(VHH) sequence in TCP021-P: QVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSS (Sequence ID 31) GFP scFv sequence in TCP001-N: QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS (Sequence ID 32) Anti-TGFβscF(mAb12.7)-US7494651B2: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSEWMNWVRQAPGQGLEWMGQIFPALGSTNYNEMYEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGIGNYALDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDFYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNIEDPLTFGGGTKVEIK (Sequence ID 33) PD-L1 BMK1 VHH (Embafolimab) - US20180327494: QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS (Sequence ID 34) 1444(VHH) protein sequence: QVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTGMYYCAASNKDYNDYDPDWGQGTQVTVSS (Sequence ID 35) B2121 scFv array in TCP001-P: DIVLTQSPASLAMSLGERATISCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISRVQAEDAAIYSCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGESVKISCKASGYTFTDYSINWVKQAPGQGLKWMGWINTETREPAYAYDFRGRFVFSLDTSASTAYLQISSLKAEDTAVYFCALDYSYAMDYWGQGTLVTVSS (Sequence ID 36) TCP001-C: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(Sequence ID 37) TCP001-P: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERC TDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAMSLGERATISCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS GSGTDFTLTISRVQAEDAAIYSCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGESVKISCKASGYTFTDYSINWVKQAPGQGLKWMGWINTETREPAYAYDFRGRFVFSLDTSASTAYLQISSLKAEDTAVYFCALDYSYAMDYWGQGTLVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(Sequence ID 38) TCP001-N: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(Sequence ID 39) TCP011-P: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(Sequence ID 40) TCP021-P: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(SEQ ID NO: 41) TCP002-C: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSAAAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSS(SEQ ID NO: 42) TCP003-C: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSAAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHH(sequence number 43) TCP-MC: QVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(SEQ ID NO:44) TCP-MD: QLQLGAGSGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH(SEQ ID NO:45)

[0189] Exemplary embodiments of the subject matter included in this specification are shown and described, and further adaptations of the methods and systems described herein can be achieved by appropriate modifications without departing from the scope of the claims. Further, when the above methods and steps indicate specific events that occur in a particular order, the specific steps need not be performed in the order described, and the steps are intended to be performed in any order as long as the embodiments can function for the intended purpose. Therefore, as long as there are variations of the present invention that are within the spirit of the present disclosure or equivalent to the invention found in the claims, this patent is intended to cover those variations as well. Some such variations should be apparent to those skilled in the art. For example, the examples, embodiments, geometries, materials, dimensions, ratios, steps, etc. discussed above are exemplary. Therefore, the claims are not limited to the specific details of the structures and operations described in the written description and the drawings.

[0190] Embodiment Embodiment 1: An immune cell comprising an expressed immunocyte-activating factor polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular labeling domain, which secretes one or more polypeptide effector molecules.

[0191] Embodiment 2: An immune cell comprising an expressed immunocyte-activating factor polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular chimeric polypeptide comprising a binding domain or single-chain variable region fragment of a VHH antibody and a labeling domain, which secretes one or more polypeptide effector molecules.

[0192] Embodiment 3: The immune cell according to Embodiment 1 or Embodiment 2, wherein the labeling domain comprises a polypeptide derived from a structural membrane protein or fetoprotein.

[0193] Embodiment 4: An immune cell according to any one of Embodiments 1 to 3, wherein the polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators.

[0194] Embodiment 5: The immune cells according to Embodiment 4, wherein the antibody is a VHH antibody.

[0195] Embodiment 6: The immune cells according to Embodiment 4, wherein the immunomodulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFβ, TGFβR, HVEM, or LIGHT.

[0196] Embodiment 7: An immune cell according to any one of Embodiments 1 to 6, wherein the labeling domain specifically binds to a bispecific polypeptide comprising a labeling-binding domain containing a single-chain polypeptide and a cell surface protein-binding domain containing a single-chain polypeptide that binds to a cell surface receptor of the cell.

[0197] Embodiment 8: (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding amino acid sequences of immune cell activator polypeptides, including a signaling domain, a transmembrane domain, and a labeling domain; and (c) Terminator regions effective in terminating transcription in immune cells; Immune cells containing nucleic acid vectors.

[0198] Embodiment 9: (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (c) Terminator regions effective in terminating transcription in immune cells; The immune cells according to Embodiment 8, further comprising a second nucleic acid vector containing the same.

[0199] Embodiment 10: The immune cell according to embodiment 8, wherein the nucleic acid vector further comprises a polynucleotide encoding the amino acid sequence of one or more secreted polypeptide effector molecules.

[0200] Embodiment 11: The immune cell according to any one of embodiments 8 to 10, wherein the immunocyte activating factor polypeptide further comprises a binding domain of a VHH antibody or a single-chain variable region fragment.

[0201] Embodiment 12: The immune cell according to any one of embodiments 8 to 11, wherein the immunocyte activating factor polypeptide comprises a chimeric polypeptide comprising (i) a binding domain of a VHH antibody or a single-chain variable region fragment and (ii) a labeling domain.

[0202] Embodiment 13: The immune cell according to embodiment 12, wherein the chimeric polypeptide is branched.

[0203] Embodiment 14: The immune cell according to any one of embodiments 8 to 13, wherein the labeling domain comprises a polypeptide derived from fetoprotein.

[0204] Embodiment 15: The immune cell according to any one of embodiments 8 to 13, wherein the labeling domain comprises a structural membrane protein.

[0205] Embodiment 16: The immune cell according to any one of embodiments 8 to 15, wherein the signaling domain comprises a co-stimulatory domain and a T cell receptor (TCR) signaling domain.

[0206] Embodiment 17: The immune cell according to embodiment 16, wherein the co-stimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40 or CD40L.

[0207] Embodiment 18: The immune cell according to embodiment 16 or embodiment 17, wherein the TCR signaling domain comprises CD3ζ or CD3ε.

[0208] Embodiment 19: An immune cell according to any one of Embodiments 16 to 18, wherein the signaling domain includes CD28 and CD3ζ.

[0209] Embodiment 20: An immune cell according to any one of Embodiments 8 to 19, wherein the transmembrane domain includes a domain involved in immune costimulatory signaling.

[0210] Embodiment 21: An immune cell according to any one of Embodiments 8 to 20, wherein the transmembrane domain includes CD28.

[0211] Embodiment 22: The immune cell according to Embodiment 21, wherein CD28 contains an ITAM domain.

[0212] Embodiment 23: An immune cell according to any one of Embodiments 8-18 and 20-22, wherein the CD3ε domain contains the amino acid YMNM.

[0213] Embodiment 24: An immune cell according to any one of Embodiments 8 to 23, wherein at least one nucleic acid vector further comprises PiggyBac transposase.

[0214] Embodiment 25: An immune cell according to any one of Embodiments 8 to 23, wherein at least one nucleic acid vector further comprises a transposon reverse-terminal repeat sequence.

[0215] Embodiment 26: An immune cell according to any one of Embodiments 8 to 25, wherein the polypeptide effector molecule comprises an antibody or a conjugated fragment thereof that specifically binds to one or more immunomodulators.

[0216] Embodiment 27: The immune cells according to Embodiment 26, wherein the antibody is a VHH antibody.

[0217] Embodiment 28: Immune cells according to Embodiment 26 or 27, wherein the immunomodulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFβ, TGFβR, HVEM, or LIGHT.

[0218] Embodiment 28: An immune cell according to any one of Embodiments 8 to 25, wherein the polypeptide effector molecule contains a cytokine.

[0219] Embodiment 30: The immune cell according to Embodiment 29, wherein the cytokine is TGF-β, VEGF, TNF-α, CCR5, CCR7, IL-2, IL-7, IL-15, or IL-17.

[0220] Embodiment 31: An immune cell according to any one of Embodiments 8 to 30, which is a T cell, a tumor-infiltrating lymphocyte, a cytokine-activated killer cell, a dendritic cell-cytokine-activated killer cell, a γδ-T cell, a natural killer T cell, or a natural killer cell.

[0221] Embodiment 32: (a) Label domain; (b) Transmembrane domain; and (c) Signal transduction domain A polypeptide containing immune cell activators.

[0222] Embodiment 33: The immune cell activator polypeptide according to Embodiment 32, wherein the signaling domain comprises a co-stimulatory domain and a T cell receptor (TCR) signaling domain.

[0223] Embodiment 34: The immune cell activator polypeptide according to Embodiment 33, wherein the co-stimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L.

[0224] Embodiment 35: The immune cell activator polypeptide according to Embodiment 33, wherein the TCR signaling domain comprises CD3ζ or CD3ε.

[0225] Embodiment 36: The immune cell activator polypeptide according to Embodiment 33, wherein the signaling domain comprises CD28, whose C-terminus is linked to the N-terminus of the CD3ε signaling domain.

[0226] Embodiment 37: The immune cell activator polypeptide according to Embodiment 33, wherein the signaling domain comprises a costimulatory domain 4-1BB, whose C-terminus is linked to the N-terminus of the CD3ε signaling domain.

[0227] Embodiment 38: An immune cell activator polypeptide according to any one of Embodiments 32 to 37, wherein the labeled domain comprises a polypeptide derived from fetoprotein.

[0228] Embodiment 39: An immune cell activator polypeptide according to any one of Embodiments 32 to 37, wherein the labeled domain comprises a structural membrane protein.

[0229] Embodiment 40: An immune cell activator polypeptide according to any one of Embodiments 32 to 39, wherein the transmembrane domain includes a domain involved in immune costimulatory signaling.

[0230] Embodiment 41: An immune cell activator polypeptide according to any one of Embodiments 32 to 40, wherein the transmembrane domain comprises CD28 or a structural membrane protein.

[0231] Embodiment 42: An immune cell activator polypeptide according to any one of Embodiments 32 to 41, wherein CD28 comprises an ITAM domain.

[0232] Embodiment 43: An immune cell activator polypeptide according to any one of Embodiments 32 to 42, wherein the CD3ε domain contains the amino acid YMNM.

[0233] Embodiment 44: (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding the amino acid sequence of an immune cell activator polypeptide; (c) Terminator regions effective in terminating transcription in immune cells; A nucleic acid vector containing [the specified ingredient].

[0234] Embodiment 45: The nucleic acid vector according to Embodiment 44, further comprising a transposon reverse-terminal repeat sequence.

[0235] Embodiment 46: (a) Promoter regions effective for transcription in immune cells; (b) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (c) Terminator regions effective in terminating transcription in immune cells; A nucleic acid vector containing [the specified ingredient].

[0236] Embodiment 47: The nucleic acid vector according to Embodiment 46, further comprising a transposon reverse-terminal repeat sequence.

[0237] Embodiment 48: A nucleic acid vector according to Embodiment 46 or Embodiment 47, wherein the polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators.

[0238] Embodiment 49: The nucleic acid vector according to Embodiment 48, wherein the antibody is a VHH antibody.

[0239] Embodiment 50: A nucleic acid vector according to Embodiment 46 or Embodiment 47, wherein the polypeptide effector molecule contains a cytokine.

[0240] Embodiment 51: (a) A label-binding domain (L-bd) comprising a single-strand polypeptide domain that specifically binds to the label domain of an immune cell activator polypeptide described in any one of Embodiments 32 to 40; and (b) A cell surface protein binding domain (CSP-bd) containing a single-chain polypeptide domain that binds to cell surface receptors on cells. A bispecific polypeptide containing [the specified component].

[0241] Embodiment 52: The bispecific polypeptide according to Embodiment 51, wherein the label-binding domain contains the VHH domain of camelid IgG.

[0242] Embodiment 53: A bispecific polypeptide according to Embodiment 51 or Embodiment 52, comprising approximately 15 to 20 amino acids of the CDR3 domain.

[0243] Embodiment 54: A bispecific polypeptide according to any one of Embodiments 51 to 53, wherein the cell is a lymphocyte.

[0244] Embodiment 55: The bispecific polypeptide according to Embodiment 54, wherein the lymphocytes are B cells.

[0245] Embodiment 56: A bispecific polypeptide according to any one of Embodiments 51 to 53, wherein the cells are tumor cells.

[0246] Embodiment 57: The bispecific polypeptide according to Embodiment 56, wherein the tumor is lymphoma, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, or mesothelioma.

[0247] Embodiment 58: The bispecific polypeptide according to Embodiment 56 or 57, wherein the cell surface protein is EGFR.

[0248] Embodiment 59: The bispecific polypeptide according to Embodiment 56 or 57, wherein the cell surface protein is GPC3.

[0249] Embodiment 60: A bispecific polypeptide according to any one of Embodiments 51 to 57, wherein the cell surface protein-binding domain specifically binds to the EGFR protein expressed on the surface of tumor cells.

[0250] Embodiment 61: A bispecific polypeptide according to any one of Embodiments 51 to 57, wherein the cell surface protein-binding domain specifically binds to CD19, CD20, or CD22 on the surface of lymphoma cells.

[0251] Embodiment 62: A bispecific polypeptide according to any one of Embodiments 51 to 57, comprising a VHH antibody.

[0252] Embodiment 63: A bispecific polypeptide according to any one of Embodiments 51 to 62, further comprising one or more domains that provide additional biochemical activity or biological function.

[0253] Embodiment 64: The bispecific polypeptide according to Embodiment 63, wherein additional biochemical activity or biological function includes specific binding of a fluorescent dye, extension of the bispecific polypeptide half-life in vivo, increased affinity of the bispecific polypeptide, and modulation of the immune response mediated by the Fc domain.

[0254] Embodiment 65: A bispecific polypeptide according to any of Embodiments 51 to 62, comprising an additional cell surface protein-binding domain(s) containing a single-chain polypeptide domain(s) that binds to different cell surface receptors(s) on the same or different cells.

[0255] Embodiment 66: A kit for in situ production of one or more polypeptide effector molecules proximal to target cells, (a) Immune cells according to any one of embodiments 8 to 31; and (b) Bispecific polypeptide according to any one of embodiments 51 to 66 A kit that includes this.

[0256] Embodiment 67: The kit according to Embodiment 66, wherein the cell surface protein-binding domain specifically binds to CD19 on B cells.

[0257] Embodiment 68: The kit according to Embodiment 66 or Embodiment 68, wherein the cell surface protein-binding domain specifically binds to EGFR, mesoserine, BCMA, MUC1, or GPC3 on tumor cells.

[0258] Embodiment 69: A method for modulating the local immune system environment of tumor cells in a subject, comprising: (a) A step of simultaneously or sequentially administering an effective amount of immune cells described in any one of Embodiments 9 to 31 and an effective amount of a first bispecific polypeptide described in any one of Embodiments 51 to 65, wherein the bispecific polypeptide comprises a cell surface protein-binding domain that specifically binds to the cell surface proteins of lymphocytes, and (b) A step of administering an effective amount of a second bispecific polypeptide described in any one of embodiments 51 to 65, wherein the bispecific polypeptide comprises a cell surface protein binding domain that specifically binds to cell surface proteins of tumor cells. A method that includes this.

[0259] Embodiment 70: The method according to Embodiment 69, further comprising the step of measuring the amount of immune cells in a subject, which is performed between steps a and b.

[0260] Embodiment 71: The method according to Embodiment 70, wherein the amount of immune cells in the target blood is measured.

[0261] Embodiment 72: The method according to Embodiment 70, wherein the amount of immune cells infiltrating the target tumor is measured.

[0262] Embodiment 73: The method according to any one of Embodiments 69 to 72, wherein the immune cells are T cells, tumor-infiltrating lymphocytes, cytokine-activated killer cells, dendritic cell-cytokine-activated killer cells, γδ-T cells, natural killer T cells, or natural killer cells.

[0263] Embodiment 74: The method according to any one of Embodiments 69 to 73, wherein the cell surface protein of the lymphocyte is CD19 of the B cell.

[0264] Embodiment 75: The method according to any one of Embodiments 69 to 74, wherein the tumor cells are lymphoma cells, mesothelioma cells, non-small cell lung cancer cells, ovarian cells, liver cancer cells, or breast cancer cells.

[0265] Embodiment 76: The method according to Embodiment 75, wherein the cell surface protein is EGFR, mesoserine, BCMA, MUC1, or GPC3.

[0266] Embodiment 77: A method for regulating the local immune system environment of tumor cells in a subject, (a) A step of growing target transformed immune cells in vitro to obtain proliferated T cells, wherein the immune cells have (i) a promoter region that is effective for transcription in the immune cells; (ii) Polynucleotides encoding the amino acid sequence of an immune cell activator polypeptide; (iii) comprising a first nucleic acid vector containing a terminator region effective for terminating transcription in immune cells; (iv) Promoter regions effective for transcription in immune cells; (v) Polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; (vi) Terminator regions effective in terminating transcription in immune cells; A step further comprising a second nucleic acid vector containing ; and a step of administering to the proliferated T cells; Furthermore (c) A step of administering to the target an effective amount to activate the proliferated immune cells and express a bispecific polypeptide immunomodulatory polypeptide comprising a label-binding domain of a determined amino acid sequence that specifically binds to a label domain expressed by the proliferated immune cells and a cell surface protein-binding domain that specifically binds to a cell surface receptor of tumor cells. Methods that include...

[0267] Embodiment 78: The method according to Embodiment 77, wherein the tumor cells are mesothelioma cells overexpressing mesoserine and PDL1, the cell surface protein is mesoserine expressed on the surface of mesothelioma cells, and the effector molecule comprises a VHH domain that specifically binds to PD-1 or CD40.

[0268] Embodiment 79: The method according to Embodiment 77 or Embodiment 78, wherein the tumor cells are B cells and the cell surface proteins are CD19, CD20, or CD22 on the surface of the B cells.

[0269] Embodiment 80: The method according to any one of Embodiments 77 to 79, wherein the immune cells are T cells, tumor-infiltrating lymphocytes, cytokine-activated killer cells, dendritic cell-cytokine-activated killer cells, γδ-T cells, natural killer T cells, or natural killer cells.

Claims

1. Immune cells comprising an expressed immune cell activator polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular labeling domain, wherein the extracellular labeling domain specifically binds to a bispecific polypeptide comprising (i) a labeling-binding domain comprising a single-chain polypeptide, and (ii) a cell surface protein-binding domain comprising a single-chain polypeptide that binds to a cell surface protein of a target cell.

2. The immune cell according to claim 1, wherein the cell surface protein of the target cell is CD19, BCMA, or EGFR.

3. The immune cell according to claim 1, wherein the target cell is a lymphocyte and the cell surface protein of the lymphocyte is CD19.

4. The immune cell according to claim 1, wherein the target cell is a tumor cell.

5. The immune cells according to claim 4, wherein the tumor cells are lymphoma cells, mesothelial cells, non-small cell lung cancer cells, ovarian cancer cells, liver cancer cells, or breast cancer cells.

6. The immune cell according to claim 4, wherein the cell surface protein of the tumor cell is CD19, CD20, CD22, EGFR, BCMA, MUC1, or GPC3.

7. The immune cell according to any one of claims 1 to 6, wherein the immune cell secretes one or more polypeptide effector molecules.

8. The polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators, wherein the antibody is a VHH antibody, or The polypeptide effector molecule contains cytokines, The immune cell according to claim 7.

9. The immune cells according to claim 8, wherein the immunomodulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFβ, TGFβR, HVEM, or LIGHT.

10. The immune cell according to claim 8, wherein the cytokine is TGF-β, VEGF, TNF-α, CCR5, CCR7, IL-2, IL-7, IL-15, or IL-17.

11. The signal transduction domain comprises a costimulatory domain and a T cell receptor (TCR) signal transduction domain, wherein the costimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L, and the TCR signal transduction domain comprises CD3ζ or CD3ε, or The signal transduction domain comprises CD28 and CD3ζ, and the CD3ε domain comprises the amino acid YMN. The immune cell according to any one of claims 1 to 10.

12. The transmembrane domain includes a domain involved in immune costimulatory signaling, and / or the transmembrane domain includes CD28, and the CD28 includes an ITAM domain. The immune cell according to any one of claims 1 to 10.

13. An immune cell according to any one of claims 1 to 12, which is a T cell, a tumor-infiltrating lymphocyte, a cytokine-activated killer cell, a dendritic cell-cytokine-activated killer cell, a γδ-T cell, a natural killer T cell, or a natural killer cell.

14. An immune cell according to any one of claims 1 to 13, (a) A label-binding domain comprising a single-chain polypeptide domain that specifically binds to the extracellular label domain of the immune cell activator polypeptide of the immune cell; and (b) A cell surface protein binding domain comprising a single-chain polypeptide domain that binds to the cell surface protein of the target cell. A bispecific polypeptide containing A kit that includes this.

15. The kit according to claim 14, wherein the bispecific polypeptide comprises a VHH antibody.

16. The kit according to claim 14, wherein the label-binding domain of the bispecific polypeptide comprises the VHH domain of a camelid IgG.

17. The kit according to any one of claims 14 to 16, wherein the bispecific polypeptide comprises 15 to 20 amino acids of the CDR3 domain.

18. (a) The target cell is a lymphocyte, and the cell surface protein of the lymphocyte is CD19, or (b) The target cells are tumor cells, and the tumor cells are lymphoma cells, mesothelial cells, non-small cell lung cancer cells, ovarian cancer cells, liver cancer cells, or breast cancer cells. The kit according to any one of claims 14 to 17.

19. The kit according to claim 18, wherein the cell surface protein of the tumor cell is CD19, CD20, CD22, EGFR, BCMA, MUC1, or GPC3.

20. The kit according to claim 14, wherein the cell surface protein of the target cell is CD19, BCMA, or EGFR.

21. The kit according to any one of claims 14 to 20, wherein the bispecific polypeptide further comprises one or more domains that provide additional biochemical activity or biological function.

22. The kit according to any one of claims 14 to 21, wherein the bispecific polypeptide comprises additional cell surface protein-binding domains, each comprising a single-chain polypeptide domain that binds to different cell surface proteins of the same or different target cells.

23. The aforementioned immune cells secrete one or more polypeptide effector molecules, and the kit is intended for use in producing the one or more polypeptide effector molecules in situ near the target cells. The kit according to any one of claims 14 to 22.

24. A composition for use in a method for modulating the local immune system environment of tumor cells in a subject, wherein the composition comprises immune cells according to any one of claims 1 to 13, and the method is (a) A step of administering an effective amount of the immune cells and an effective amount of a first bispecific polypeptide comprising a label-binding domain that specifically binds to the extracellular label domain of the immune cell activator polypeptide of the immune cells and a cell surface protein-binding domain that specifically binds to the cell surface protein of the target cell, wherein the target cell is a lymphocyte, and (b) A step of administering an effective amount of a second bispecific polypeptide comprising a label-binding domain that specifically binds to the extracellular label domain of an immune cell activator polypeptide of the immune cell, and a cell surface protein-binding domain that specifically binds to a cell surface protein of the target cell, wherein the target cell is a tumor cell. A composition containing the following:

25. The composition for use according to claim 24, further comprising the step of measuring the amount of immune cells in the subject between step (a) and step (b).

26. The composition for use according to claim 24 or 25, wherein the cell surface protein of the lymphocyte is CD19.

27. The composition for use according to any one of claims 24 to 26, wherein the tumor cells are lymphoma cells, mesothelial cells, non-small cell lung cancer cells, ovarian cancer cells, liver cancer cells, or breast cancer cells.

28. The composition for use according to claim 27, wherein the cell surface protein of the tumor cell is CD19, CD20, CD22, EGFR, BCMA, MUC1, or GPC3.

29. The composition for use according to claim 24 or 25, wherein the cell surface proteins of the target cells are CD19, BCMA, and EGFR.

30. A composition for use in a method for modulating the local immune system environment of tumor cells in a subject, wherein the composition comprises immune cells according to any one of claims 1 to 13, and the method is (a) A step of growing the transformed immune cells of the subject in vitro to obtain the grown immune cells, wherein the immune cells (i) Promoter regions effective for transcription in immune cells; (ii) A polynucleotide encoding the amino acid sequence of an immune cell activator polypeptide, wherein the immune cell activator polypeptide comprises a signaling domain, a transmembrane domain, and a labeling domain comprising the sequence of SEQ ID NO: 20; and (iii) Terminator regions effective in terminating transcription in immune cells; A first nucleic acid vector, comprising a nucleic acid vector containing, and (i) Promoter regions effective for transcription in immune cells; (ii) polynucleotides encoding the amino acid sequence of one or more secreted polypeptide effector molecules; and (iii) Terminator regions effective in terminating transcription in immune cells; A second nucleic acid vector containing Steps including; (b) The step of administering the proliferated immune cells to the subject; Furthermore (c) A step of administering an effective amount of a bispecific polypeptide to the subject, comprising a label-binding domain that specifically binds to the label domain of the immune cell activator polypeptide and a cell surface protein-binding domain that specifically binds to the cell surface protein of the tumor cell, thereby activating the proliferated immune cells and expressing one or more polypeptide effector molecules. A composition containing the following:

31. The composition for use according to claim 30, wherein the tumor cells are lymphoma cells, mesothelial cells, non-small cell lung cancer cells, ovarian cancer cells, liver cancer cells, or breast cancer cells.

32. The composition for use according to claim 30, wherein the effector molecule comprises a VHH domain that specifically binds to PD-1 or CD40.

33. The composition for use according to any one of claims 24 to 32, wherein the immune cells are T cells, tumor-infiltrating lymphocytes, cytokine-activated killer cells, dendritic cell-cytokine-activated killer cells, γδ-T cells, natural killer T cells, or natural killer cells.

34. The immune cell according to claim 1, the kit according to claim 14, or the composition for use according to claim 24 or claim 30, wherein the label-binding domain comprises the sequence of SEQ ID NO: 28.

Images