Cells expressing immunomodulatory molecules and systems expressing immunomodulatory molecules

Abstract

Disclosed herein are immune cells (Bisuper Cells, BS-Cells) that have been engineered to express an immune cell activator polypeptide comprising an extracellular marker domain and incorporate the polypeptide into the cell membrane surface. Also disclosed are immune cells engineered to secrete one or more polypeptide effector molecules, and 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 immune cell activator polypeptides to another cell. Also disclosed are systems comprising both immune cells and various bispecific polypeptides that can bind to different cell surface proteins of the same or different target cells, e.g., that can be used to expand immune cells in vivo and treat various tumors.

Description

TECHNICAL FIELD

[0001] The subject matter disclosed herein relates to cells expressing immune system modulatory proteins or other effector polypeptides, chimeric immune cell activator polypeptides (ICAPs), herein referred to as "white blood super cells," and to systems for controlling the expression of these proteins and polypeptides in these cells. Such systems can include polypeptides having bi-specific binding activity, and thus can activate cells bearing vectors expressing immune system modulatory proteins or other effector polypeptides upon binding to a polypeptide target domain. BACKGROUND

[0002] T cells bearing chimeric antigen receptors (CAR-T cells) are being developed as an immunotherapeutic modality for cancer treatment. Typically, CARs include an extracellular domain that binds an activating ligand, a transmembrane domain that participates in the formation of an immunological synapse with a "target" cell, and an intracellular domain that responds to extracellular domain binding by activating T cell-associated transcriptional responses.

[0003] Current CAR-T cell-based therapies are ineffective against tumors with heterogeneous TAA expression or variants that have lost emerging antigens due to having a single TAA (tumor associated antigen) recognition extracellular domain in the CAR.

[0004] Current CAR-T cell-based therapies rely on ex vivo proliferation of CAR-T cells prior to treatment of the patient.

[0005] Furthermore, there is no simple method available for monitoring CAR-T cell distribution and fate in vivo.

[0006] Without any method of controlling the activity of activated CAR-T cells or methods of depleting unwanted CAR-T cells, other CAR-T cells continue to proliferate and activate in response to antigen uncontrolled, potentially leading to fatal off-target toxicities, cytokine release syndrome or neurotoxicity.

[0007] Most CAR extracellular antigen recognition domains are scFv proteins, and two scFv domains can form a non-covalently linked dimer, for example by domain swapping. This type of interaction between adjacent scFv domains greatly enhances tonic signaling in CAR-T cells, leading to uncontrolled activity. SUMMARY

[0008] Disclosed herein are immune cells that have been engineered to express an immune cell activator polypeptide (ICAP) and incorporate the polypeptide into the cell membrane surface. Also disclosed are immune cells engineered to secrete one or more polypeptide effector molecules, and immune cells engineered to express two molecules.

[0009] Accordingly, in one aspect of the disclosure, there is provided an immune cell comprising a nucleic acid vector (or a first nucleic acid vector), the nucleic acid vector comprising

[0010] (a) a promoter region effective for transcription in an immune cell;

[0011] (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0012] (c) a terminator region effective to terminate transcription in an immune cell.

[0013] Such an immune cell can be a cell further comprising a second nucleic acid vector, the second nucleic acid vector comprising

[0014] (d) a promoter region effective for transcription in an immune cell;

[0015] (e) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; and

[0016] (f) a terminator region effective to terminate transcription in an immune cell.

[0017] Alternatively, the engineered immune cell can be an immune cell wherein the first nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.

[0018] Another aspect of the disclosure relates to an immune cell activator polypeptide comprising:

[0019] (a) a marker domain;

[0020] (b) a transmembrane domain; and

[0021] (c) a signal transduction domain.

[0022] Another aspect of the disclosure is a nucleic acid vector comprising

[0023] (a) a promoter region effective for transcription in an immune cell;

[0024] (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0025] (c) a terminator region effective to terminate transcription in an immune cell.

[0026] Another aspect of the disclosure is a nucleic acid vector comprising

[0027] (a) a promoter region effective for transcription in an immune cell;

[0028] (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.

[0029] (c) a terminator region effective to terminate transcription in the immune cell.

[0030] Another aspect of the disclosure is a bispecific polypeptide that is a nanobody targeting a target cell and controlling an immune cell (VHH-TCP), comprising:

[0031] (a) a label-binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a label domain of an immune cell activator polypeptide; and

[0032] (b) a cell surface protein-binding domain (CSP-bd) comprising a single chain polypeptide domain that specifically binds to a cell surface receptor of a cell.

[0033] The disclosure also describes a kit for producing one or more polypeptide effector molecules in situ in the vicinity of a target cell, comprising:

[0034] I. an immune cell comprising a nucleic acid vector comprising

[0035] (a) a promoter region effective for transcription in the immune cell;

[0036] (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide, the polypeptide comprising a signal transduction domain, a transmembrane domain, and a label domain; and

[0037] (c) a terminator region effective to terminate transcription in the immune cell, and

[0038] a second nucleic acid vector comprising

[0039] (a) a promoter region effective for transcription in the immune cell;

[0040] (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules;

[0041] (c) a terminator region effective to terminate transcription in the immune cell;

[0042] and

[0043] II. a bispecific polypeptide comprising:

[0044] (a) a label-binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a label domain of an immune cell activator polypeptide; and

[0045] (b) a cell surface protein-binding domain (CSP-bd) comprising a single chain polypeptide domain that specifically binds to a cell surface receptor of a cell.

[0046] Alternatively, the engineered immune cell can be one in which the first nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of a secreted effector polypeptide. In such embodiments, the secreted effector polynucleotide can be encoded in a second expression cassette. Such kits further comprise a bispecific polypeptide that is a targeted and controlled nanobody polypeptide (VHH-TCP) comprising:

[0047] (a) a label-binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a label domain of the immune cell activator polypeptide; and

[0048] (b) a cell surface protein-binding domain (CSP-bd) comprising a single chain polypeptide domain that binds to a cell surface receptor of a cell.

[0049] The present disclosure also provides a method of modulating an immune system environment at a location of a tumor cell in a subject, the method comprising:

[0050] (a) administering to the subject an effective amount of an engineered immune cell comprising a first nucleic acid vector, the first nucleic acid vector comprising:

[0051] (i) a promoter region effective for transcription in an immune cell;

[0052] (ii) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0053] (iii) a terminator region effective to terminate transcription in the immune cell.

[0054] and further comprising a second nucleic acid vector, the second nucleic acid vector comprising

[0055] (i) a promoter region effective for transcription in an immune cell;

[0056] (ii) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; and

[0057] (iii) a terminator region effective to terminate transcription in the immune cell.

[0058] (b) simultaneously or sequentially administering to the subject an effective amount of a first bispecific polypeptide, the polypeptide comprising:

[0059] (i) a label-binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a label domain of the immune cell activator polypeptide; and

[0060] (ii) a cell surface protein-binding domain (CSP-bd) comprising a single chain polypeptide domain that specifically binds to a cell surface protein of a lymphocyte.

[0061] (c) administering to the subject an effective amount of a second bispecific polypeptide, the polypeptide comprising:

[0062] (i) a marker binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a marker domain of the immune cell activator polypeptide; and

[0063] (ii) a cell surface protein binding domain (CSP-bd) comprising a single chain polypeptide domain that specifically binds to a cell surface protein of a tumor cell.

[0064] A step of measuring the number of engineered immune cells in the subject can be performed between steps b and c.

[0065] The method of modulating the immune system environment at the location of tumor cells in a subject can alternatively comprise:

[0066] (a) proliferating transformed T cells of the subject in vitro, wherein the T cells comprise a first nucleic acid vector, the first nucleic acid vector comprising a nucleic acid vector comprising:

[0067] (i) a promoter region operable for transcription in an immune cell;

[0068] (ii) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0069] (iii) a terminator region operable to terminate transcription in an immune cell;

[0070] and comprising a second nucleic acid vector, the second nucleic acid vector comprising

[0071] (i) a promoter region operable for transcription in an immune cell;

[0072] (ii) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; and

[0073] (iii) a terminator region operable to terminate transcription in an immune cell;

[0074] to obtain the proliferated T cells; and administering the proliferated T cells to the subject; and

[0075] (b) administering to the subject an effective amount to activate the proliferated T cells to express a secreted polypeptide effector molecule VHH-TCP, the VHH-TCP comprising a CSP-bd and an L-bd having a determined amino acid sequence that specifically binds to a marker domain expressed by the proliferated T cells, the CSP-bd specifically binds to a cell surface protein of a tumor cell. SUMMARY

[0077] While the specification concludes with claims particularly pointing out and distinctly claiming that which is described, the specification is believed to be better understood in conjunction with the accompanying drawings and following description in which like reference characters refer to like elements and in which:

[0078] Figure 1 Depicted are exemplary effector cells described herein, e.g.,“white super cells,” which show expression of immune cell activator polypeptides and secrete immunomodulatory effector molecules; shown is an anti-PD1-VHH nanobody.

[0079] Figure 2 Described are nanobody targeting and control polypeptides (VHH-TCP); shown are domains that bind to a marker domain of an immune cell activator protein (Marker-VHH) and a cell surface protein of a cell, here CD19 ligand of a B cell (CD19-VHH). Also shown are other domains for activating Fc-mediated immune responses (hFc-VHH), FITC fluorophore binding (FITC-VHH), and binding to serum albumin (Albumin-VHH).

[0080] Figure 3A and 3B Depicted are exemplary expression vector maps for expressing ICAPs and effector proteins in immune cell-like host cells. In Figure 3A the effector protein anti-PD-1-VHH-Fc(EQ) is expressed from a structural gene in an expression construct in vector pS338B-1182-Fc(EQ). In Figure 3B the ICAP with marker polypeptide domain, CD8 hinge domain, CD28 transmembrane (TM) domain, and CD28 intracellular signaling domain and CD3z domain is expressed from a structural gene in expression construct pNB338B-ICAPs-VHH.

[0081] Figure 4 A depicts flow binding affinity of M 2339 (VHH) to mesothelin (MSLN). Figure 4 B shows flow binding affinity of B029 (VHH) to BCMA. Figure 4 C shows flow binding affinity of E454 (VHH) to EGFR.

[0082] Figure 5 Shown are binding kinetics of M2339 VHH-6his to different mesothelin ECD domains determined by surface plasmon resonance (SPR).

[0083] Figure 6Figures showing various M-ICAP (derived from the mesothelin II+III region) vectors. 19R73 is a version of the classic CD19 CAR-T (positive control), the others are M-ICAP vectors. The intracellular region of these vectors is the same, all containing 4-BB and CD3 zeta, but the extracellular region is different. M-ICAP does not contain a 6-His tag. His-1 / 2-M-ICAP: 6-His tag at the N- or C-terminus of M-ICAP. SP3-His-M-ICAP and SP5 His-M-ICAP: signal peptide is SP3 or SP5, selected from the human protein database. SP3 (signal peptide 3): MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 1); SP5 (signal peptide 5): MTRLTVLALLAGLLASSRA (SEQ ID NO: 2).

[0084] Figure 7 FACS results of M-ICAP vectors transfected into 293T cells are shown. Figure 7 A shows the positive rate of 293T cells transfected with different M-ICAP vectors. Figure 7 B shows dot plots of FACS results.

[0085] Figure 8 M-ICAP expression and M-ICAP-T cell construction are illustrated. Figure 8 A is a schematic of M-ICAP-T cell construction. Figure 8 B provides a schematic of M-ICAP, SP3-M-ICAP and SP5-M-ICAP expression vectors. Figure 8 C and 8D present positive rate data for M-ICAP, SP3-M-ICAP and SP5-M-ICAP T cells 8 and 13 days post transfection Figure 8 C: activated by M2339 + anti-CD28 or anti-His + anti-CD28, respectively; Figure 8 D: activated by M2339 + anti-CD28). Abbreviations: M-ICAP - peptide derived from mesothelin II+III; SP - endogenous signal peptide of mesothelin; SP3 (signal peptide 3): MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 1); SP5 (signal peptide 5): MTRLTVLALLAGLLASSRA (SEQ ID NO: 2); M2339, anti-M-ICAP - VHH-Fc clone M2339; anti-CD28 - anti-CD28 monoclonal antibody; anti-His - anti-His mAb.

[0086] Figure 9 Representative preparations of ICAP-T cells and quality verification are shown. Figure 9A provides a schematic of M-ICAP, M-ICAP-28, M-ICAP-28BB expression vectors. Figure 9 B shows a comparison of ICAP-T expansion (from peripheral blood mononuclear cells, PBMC) obtained by different TCPs or antibodies activation. Figure 9 C shows expansion during ICAP-T cell preparation from PBMC. Figure 9 D shows the ICAP positive proportion of ICAP-T cell product. Figure 9 E shows the CD4 / CD8 positive proportion in CD3 positive cells of ICAP-T cell product. Figure 9 F shows the Tem / Tcm positive proportion in Tm cells of ICAP-T cell product.

[0087] Figure 10 Binding affinity of BCMA-TCPs measured by FACS is shown. Figure 10 A shows FACS binding curves of three BCMA-TCPs to cells of MSLN overexpressing cell line. Figure 10 B shows FACS binding curves of three BCMA-TCPs to cells of BCMA overexpressing cell line.

[0088] Figure 11 Plasma stability of anti-BCMA TCPs is shown.

[0089] Figure 12 Binding affinity of TCP011-P to two different cell types measured by FACS is shown. Figure 12 A shows FACS binding curves of TCP011-P to cells of CD19 overexpressing cell line. Figure 12 B shows FACS binding curves of TCP011-P to cells of MSLN overexpressing cell line.

[0090] Figure 13 Binding affinity of TCP021-P to two different cell types measured by FACS is shown. Figure 13 A shows FACS binding curves of TCP021-P to EGFR overexpressing cells. Figure 13 B shows FACS binding curves of TCP021-P to MSLN overexpressing cells.

[0091] Figure 14 In vitro expansion of M-ICAP-T with TCPs to target cells is shown. Figure 14 A and 14B show T / Daudi cell counts after 4 days of co-culture of M-ICAP transfected T and Daudi cells with TCPs. Figure 14C and 14D show T / Daudi cell counts after 4 days co-culture of M-ICAP transfected T and Mitomycin C (MMC) treated Daudi cells with TCP.

[0092] Figure 15 M-ICAP-T shows TCP dose dependent cytotoxic effect on RPMI-8226 cells. Figure 15 A shows a schematic of the suspension cell lysis assay. Figure 15 B shows dose dependent cell lysis release of TCP001-C of M-ICAP-T with RPMI-8226 cells at three different E:T ratios. Figure 15 C-15E shows cell lysis analysis curves at different E:T ratios.

[0093] Figure 16 ICAP / CAR-T with different TCP combinations were compared for cytotoxicity and IFNy secretion on RPMI-8226 / L363 cells. Figure 16 A and 16B compare the cytotoxic effect of ICAP / CAR-T cells with different TCP combinations on L363 cells at 0.5 (A) or 0.2 (B) ug / ml concentration. Figure 16 C and 16D show IFNy secretion of ICAP / CAR-T with different TCP combinations on RPMI-8226 (C) or L363 (D) cells.

[0094] Figure 17 ICAP with TCP (binding EGFR) combinations show cell lysis effect on FaDu / SK-OV3 cells. Figure 17 A shows cell lysis of ICAP / CAR-T cells with different TCP combinations on FaDu cells. Figure 17 B shows cell lysis of ICAP / CAR-T cells with different TCP combinations on SK-OV3 cells.

[0095] Figure 18 ICAP-T cells with TCP show IFN-g release and cell lysis effect on Daudi cells. Figure 18 A shows IFN-g release of ICAP / CAR-T cells with different TCP on Daudi cells. Figure 18 B shows cell lysis of ICAP / CAR-T cells with different TCP combinations on Daudi cells.

[0096] Figure 19 M-ICAP-T shows the ability to secrete antibodies and the positivity rate is not affected. Figure 19A shows the comparison of the positive rate of secretory M-ICAP-T cells. Human naive T cells were transfected with M3 CAR and secretory plasmids of antibodies (such as anti-PD-1, anti-TGFp and anti-PD-L1) at the same time. After 13 days, there was little difference between the four experimental groups, with a positive rate of about 60-70%. M-ICAP-T cells also secreted anti-PD-1, anti-TGFp and anti-PD-L1 antibodies well. These data show that the type and level of antibody secretion have little effect on the positive transformation of M-ICAP-T cells. VHH or scFv can be well secreted from M-ICAP-T cells and detected by ELISA.

[0097] Figure 20 It is shown that anti-PD-1-M-ICAP-T cells can secrete anti-PD-1 VHH to block surface PD-1 protein. Cells were stimulated with 5ug / ml M2339-IgG4 or IgG4 control for 48 hours. Commercial PD-1 mAb only blocks the detection of surface PD1 in the anti-PD-1 VHH M-ICAP-T cell group.

[0098] Figure 21 It is shown that anti-TGFp scFv secreted by M-ICAP-T cells binds to TGFpRII. TGFp ligand binds to TGFpRII and stimulates luciferase signal. Anti-TGFp scFv secreted by M-ICAP-T cells can also bind to TGFpRII on 293T cells and block the expression of luciferase reporter gene. CAR-T-10C, 10B and 01A are anti-TGFp M-ICAP-T cells prepared from different donors.

[0099] Figure 22 It is shown that the body weight change of L363-PDL1 orthotopic tumor in NPSG mice in the in vivo efficacy test of M-ICAP-T cells combined with TCP001-C.

[0100] Figure 23 It is shown that the tumor volume change of L363-PDL1 orthotopic tumor in NPSG mice in the in vivo efficacy test of M-ICAP-T cells combined with TCP001-C.

[0101] Figure 24 It is shown that the analysis of anti-PD-1 VHH and TCP001-C concentrations in mouse whole blood. Figure 24 A shows the analysis of serum anti-PD-1 VHH levels. Figure 24 B shows the analysis of serum TCP001-C levels. Abbreviations: D15-24h, D15 tail vein bleeding, 24h after injection of TCP001-C on day 14; D22-48h, D22 tail vein bleeding, 48h after injection of TCP001-C on day 20.

[0102] 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. The expression constructs both contain 5' and 3' ITR sequences.

[0103] Figure 25 Binding of anti-MSLN-1444 VHH (1444(VHH)) on HEK293T-MSLN cells is shown by FACS analysis.

[0104] Figure 26 Expression of fusion polypeptide BCMA ICAP BCMAmutl with anti-MSLN-1444 VHH is shown.

[0105] Figure 27 SPR kinetics of BCMA ICAP BCMAmutl binding with different anti-BCMA VHH is shown.

[0106] Figure 28 In vitro activation and expansion of BCMAmutl-MSLN-1444 CAR-T is shown. Figure 28 A shows a schematic of the BCMAmutl-MSLN-1444 vector; Figure 28 B, expansion of BCMAmutl-MSLN-1444 CAR-T in donor 1 stimulated with anti-BCMAmutl VHH 36# or anti-MSLN; and Figure 28 C, expansion of BCMAmutl-MSLN-1444 CAR-T in donor 2 stimulated with anti-BCMAmutl VHH 36# or anti-MSLN.

[0107] Figure 29 Dot plots showing FACS results of expansion of BCMAmutl-MSLN-1444 CAR-T in 2 donors stimulated with anti-BCMAmutl VHH 36# or anti-MSLN are shown.

[0108] Figure 30 Anti-BCMAmuc1 VHH 36# specific activation and expansion of MSLN-1444 CAR-T is shown. Figure 30 A shows a schematic of the MSLN-1444 vector. Figure 30 B shows expansion of MSLN-1444 CAR-T in donor 1 stimulated with anti-BCMAmuc1 VHH 36# or anti-MSLN. Figure 30 C shows expansion of MSLN-1444 CAR-T in donor 2 stimulated with anti-BCMAmuc1 VHH 36# or anti-MSLN. DETAILED DESCRIPTION

[0109] Chimeric antigen receptor T cell (CAR-T) therapy technology belongs to the field of cancer immunocyte therapy. CAR-T technology uses genetic engineering technology to splice the intracellular region of T lymphocyte immune receptors with, for example, an antibody variable region gene sequence containing at least a portion of a gene encoding the CDR portion of an antibody, and then introduce the spliced construct into T cells through a retrovirus or lentivirus vector, transposon or transfection. The expression cassette or mRNA is transduced into lymphocytes, and a fusion protein is expressed on the cell surface, enabling T lymphocytes to recognize specific antigens in a non-MHC restricted manner, improving their ability to recognize and kill tumors.

[0110] The structure of the chimeric antigen receptor (CAR) was proposed by the Eshhar research group in Israel in 1989. Since then, T cells with cell surface proteins structured with CAR have been shown to have good effects in tumor immunotherapy.

[0111] The first generation of CAR receptors contains a single-chain variable fragment (scFv), and the intracellular activation signal is transmitted through a CD3 zeta (CD3z) signal chain. However, the first generation of CAR receptors lacks a domain that provides a T cell costimulatory signal, which results in CAR-T cells only playing a transient role, with short cell survival time in the body and low cytokine secretion. The second generation of CAR receptors incorporates intracellular domains of costimulatory signaling molecules, including, for example, CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulator (ICOS), DNAX-activating protein 10 (DAP10) and other domains to enhance T cell proliferation and cytokine secretion. IL-2, IFN-g and GM-CSF production is increased, thereby disrupting the immunosuppression of the tumor microenvironment, such as AICD (activation-induced cell death (AICD)).

[0112] The third generation of CAR receptors recombines a secondary costimulatory molecule, such as 4-1BB, between the costimulatory structure CD28 and the ITAM signal chain, thereby producing a three-signal CAR receptor.

[0113] Engineered CAR-T cells have better effector function and survival time in the body. Currently, the CAR structure commonly used in therapy is the second generation of CAR receptors, which can be divided into the following four parts: an antibody single-chain variable region (scFv), a hinge region, a transmembrane region and an intracellular stimulation signal transduction polypeptide. The hinge region structure of the CAR helps to form the correct conformation and form a dimer. The length and amino acid sequence characteristics of the hinge region help to determine the spatial conformation of the CAR, and also affect the ability of the CAR to bind to tumor cell surface antigens.

[0114] Malignant lymphomas are divided into two categories: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Hodgkin's lymphoma accounts for 10-15% of lymphomas, while non-Hodgkin's lymphoma is the most rapidly growing malignant tumor among patients with onset. According to the statistics of the World Health Organization, there are about 350,000 new NHL patients worldwide every year, and more than 200,000 people die. B-cell lymphoma can be seen in both Hodgkin's lymphoma and non-Hodgkin's lymphoma. At present, the clinical treatment methods of lymphoma include cytotoxic drugs (such as glucocorticoids and alkylating agents) and targeted drugs based on specific molecular targets (such as rituximab, etc.), among which the combination chemotherapy based on targeted drugs can significantly improve the response, clinical remission rate and cure rate of patients. However, a large number of lymphoma patients are not sensitive or have poor efficacy, which are "true" refractory patients. Some new treatment methods (such as cellular immunotherapy) have alleviated and prolonged the survival period of some relapsed or refractory lymphoma patients. At present, there are many kinds of CAR-T developed for hematological malignancies, including the use of anti-CD19, anti-CD20, anti-kappa light chain, anti-CD22, anti-CD23, anti-CD30, anti-CD70 and other antibody constructed CAR modified T cell therapy. Anti-tumor studies have been carried out, among which anti-CD19 and anti-CD20 monoclonal antibodies are the most commonly used antibodies.

[0115] Selecting the correct tumor antigen as a target is the key to designing safe and effective CAR-T cells. Since CD19 is only expressed in normal and malignant B cells at various stages of differentiation, but not in other non-B cells (such as hematopoietic stem cells), it is a potential target for treating B lineage tumors and a hot spot in CAR-T research. Therefore, CD19 CAR-T is widely used for malignant tumors such as acute B lymphoblastic leukemia (B-ALL), chronic B lymphoblastic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL and multiple myeloma (MM). CD19 CAR-T has been used in clinical trials for the treatment of B-cell lymphoma.

[0116] PD-1 (programmed death 1, programmed cell death receptor 1) is a member of the CD28 family of regulatory T cells, belonging to the immunoglobulin receptor superfamily. PD-1 and its ligand PD-L1 / PD-L2 play an important role in the co-inhibition and exhaustion of T cells. Their interaction inhibits the proliferation of co-stimulatory T cells and the secretion of cytokines. The expression of anti-apoptotic molecule BCL-x1 weakens the function of tumor-specific T cells, leading to the inability of some tumor patients to completely eliminate tumors. Anti-PD-1 antibodies compete with ligands PD-L1 / PD-L2 to bind to PD-1 molecules on the surface of tumor-specific T cells, thereby inhibiting the complex of PD-1 and PD-L1 / PD-L2. This in turn overcomes the inhibition of the immune microenvironment caused by the complex of PD-1 and PD-L1 / PD-L2.

[0117] The currently commercialized anti-PD-1 antibodies are nivolumab and pidilizumab. These two monoclonal antibodies have shown good clinical efficacy in solid tumors such as melanoma, colon cancer, prostate cancer, non-small cell lung cancer and renal cell carcinoma. Recent clinical studies have confirmed that PD-1 antibodies can be used for lymphoma treatment. However, there are still some unavoidable problems in the clinical application of anti-PD-1 antibodies. On the one hand, since the anti-PD-1 monoclonal antibody is administered intravenously, most patients receiving PD-1 antibody blockade will have varying degrees of drug administration side effects. Moreover, the in vitro production of anti-PD-1 monoclonal antibodies involves complex production preparation and purification processes, which are very expensive and lead to high treatment costs.

[0118] In summary, CAR-T cells have the ability to kill tumor cells and can effectively enter tumor tissues, but their activity is easily inhibited in the tumor microenvironment; PD-1 antibodies can reactivate the anti-tumor activity of T cells. However, conventional large molecule antibodies or large fragments thereof have insufficient penetration of solid tumors, systemic drugs have greater toxic side effects, and drug costs are high.

[0119] Therefore, the solution to this problem is disclosed herein, in which anti-PD-1 antibodies can be efficiently expressed by maintaining the killing toxicity of CAR-bearing immune cells (e.g., CAR-T cells), and the CAR-bearing cells express the PD-1 antibodies at a high level in or near the tumor. It is expected that this activity will increase the tumor-killing efficacy of CAR-bearing cells, while also reducing treatment costs.

[0120] The current disclosure is a system that has some features similar to CAR-T, but has more general properties. Moreover, by including an extracellular (possibly synthetic and not naturally occurring amino acid sequence) peptide molecule with dual specific binding activity, which is the binding of CAR-bearing effector cells and target cells bearing cell surface antigens, as an "immune cell activator polypeptide" (ICAP), the activity level of CAR-bearing effector cells can be adjusted by controlling the amount of ICAP available for binding to CAR. Such a system can be used to address the problem of high tonic activity exhibited by prior art CAR-T cells.

[0121] Some terms related to the present disclosure are explained below.

[0122] In the present disclosure, the term "expression cassette" refers to the entire element required for gene expression, including a promoter, a coding sequence, and a poly A tail signal sequence.

[0123] The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that encodes an amino acid sequence of a polypeptide product (e.g., a CAR, a single chain antibody, or a domain thereof). The boundaries of a coding sequence are typically determined by a ribosome binding site (for prokaryotes) just upstream of the open reading frame encoding the mRNA at its 5' end and a transcription stop sequence just downstream of the open reading frame at its 3' end. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0124] The term "Fc" (fragment, crystallizable) is a portion of a mammalian antibody that refers to the peptide located at the end of the "Y" structure handle of an antibody molecule, comprising the CH2 and CH3 domains of the constant region of the heavy chain of an antibody, is the site of many molecular and cellular interactions that can provide certain biological effects of a mammalian antibody.

[0125] The term "costimulatory molecule" refers to a molecule present on the surface of an antigen presenting cell that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. Proliferation of lymphocytes requires not only the binding of antigen, but also a signal from a costimulatory molecule. The costimulatory signal is transmitted to the T cell primarily through binding of the costimulatory molecule CD80 on the surface of an antigen presenting cell to the CD28 molecule on the surface of the T cell, while CD86 binds to the CD28 molecule on the surface of the T cell. B cells receive a costimulatory signal, which can be through a common pathogen component (e.g., LPS), or through a complement component, or through the activated antigen-specific Th cell surface protein CD40L.

[0126] The term "linker" is a polypeptide segment that links different proteins or polypeptides, the purpose of which is to maintain the spatial relationship of the linked proteins or polypeptides (e.g., by mitigating steric inhibition of ligand binding) to maintain the function or activity of the proteins or polypeptides. Exemplary linkers include glycine- and / or serine-containing linkers, and, for example, Furin 2A peptides.

[0127] The term "specifically binds" refers to the reaction of a binding protein with a ligand, such as the reaction between an antibody or antigen-binding fragment and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody-antigen affinity is characterized by a binding constant Kd of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. "Specifically recognizes" or "specifically recognizes" have similar meanings.

[0128] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is physio logically and / or pharmacologically compatible for use with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Gennaro AR, ed., 19thEdition, Mack Publishing Company, Pennsylvania, 1995, which is incorporated by reference herein in its entirety and for all purposes), including but not limited to pH adjusting agents, surfactants, adjuvants, ion strength enhancers. For example, pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween 80; ion strength enhancers include but are not limited to sodium chloride.

[0129] The term "effective amount" refers to a dosage that can achieve treatment, prevention, reduction, and / or alleviation of a disease or condition described herein in a subject.

[0130] The term "disease and / or condition" refers to the physical state of a subject associated with a disease and / or condition described herein.

[0131] The term "subject" or "patient" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., who receives a pharmaceutical composition of the present application to treat, prevent, ameliorate, and / or alleviate a disease or condition of the present application.

[0132] As used herein, a "chimeric antigen receptor" (CAR) is an artificially engineered protein that binds to a specific molecule (e.g., a tumor cell surface antigen) and stimulates proliferation programs in immune cell type effector cells. A CAR typically comprises, in order from amino terminus to carboxyl terminus, an optional signal peptide (which can be removed during localization of the CAR in the cell membrane of a host cell); a polypeptide that specifically binds to other proteins ("a marker domain"), such as an antigen-binding region of a single-chain antibody; an optional (but often present) hinge region; a transmembrane region; and an intracellular signaling region (see, e.g., Figure 1 The marker domain polypeptide can be a polypeptide derived from a natural polypeptide or a synthetic polypeptide.

[0133] In the present application, a "VHH domain" can 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, typically comprising a number of relatively conserved domains that come together upon folding of the polypeptide to form framework regions (FR regions), while variable regions come together to form a variable antigen binding domain. Thus, a VHH antibody is a type of SCA. In accordance with this terminology, the variable domain present in a naturally occurring single heavy chain antibody is also referred to herein as a "VHH domain" in order to distinguish it from a heavy chain variable domain present in a conventional 4-chain antibody (referred to herein as a "VH domain"), as well as from a light chain variable domain present in a conventional 4-chain antibody (referred to herein as a "VL domain").

[0134] The isolated single variable domain polypeptide is preferably a polypeptide that has all of the antigen binding capacity of its cognate SCA and is stable in aqueous solution.

[0135] A "single chain antibody" herein also encompasses a stable, antigen-binding single chain polypeptide comprising one or more domains derived from or analogous to domains of a mammalian antibody (or from FR or variable regions), such as a VH domain.

[0136] A "nanobody" can include a SCA or VHH antibody or one or more domains thereof, but the term is more typically used to describe an engineered polypeptide comprising one or more VHH domains, and optionally further comprising one or more FR domains, and additionally or alternatively can also include additional stabilizing domains with some other biological activity, such as binding to a fluorophore or binding to and activating an extracellular receptor.

[0137] Disclosed herein is a new cell therapy product, an engineered immune effector cell, which can be a so-called "white whale super cell" comprising a chimeric receptor that can be induced to express a secreted protein in situ in a controllable manner.

[0138] In some embodiments, the engineered immune effector cell constitutively expresses a high level of an effector polypeptide, such as a single chain anti-PD-1 antibody (VHH-PD-1). In some such embodiments, T cell proliferation activated by binding of a "marker domain" of the cell surface associated antigen of the immune effector cell T cell provides a very large number of T cells that constitutively secrete the effector polypeptide. In the case where the marker domain is bound by an antigen on the surface of a tumor cell, the immunomodulatory effector polypeptide can be constitutively expressed and the immune tolerance induced by, for example, formation of a PD-1 :PD-L1 / L2 complex is mitigated or avoided due to its secretion in the vicinity of the tumor cell.

[0139] Additionally or alternatively, the engineered effector cells disclosed herein can be engineered to comprise a nucleic acid vector comprising a coding sequence construct encoding one or more "effector polypeptides" expressed under the control of a promoter operable in an immune cell, and further comprising a transcription termination sequence operable in an immune cell. The promoter can be a constitutive promoter, such as an EF1a promoter or a CMV promoter.

[0140] The nucleic acid vector can be a retroviral vector or a lentiviral vector. The nucleic acid vector can be a DNA or RNA vector. The vector can comprise a PiggyBac (PB) transposon or a Sleeping Beauty (SB) transposon or a portion thereof. The vector can comprise transposon-specific inverted terminal repeat sequences, which are typically located at both ends of a transposon-based vector.

[0141] The engineered effector cells disclosed herein can be cells in which one or both of the expression cassettes encoding an ICAP and the expression cassettes encoding one or more effector polypeptides are integrated into the nuclear genome of the effector cell.

[0142] The protein to be secreted by the effector cell can be an immunostimulatory protein, such as a polypeptide that specifically binds 4-1BB or OX40, or an immunosuppressive protein (e.g., for treating an allergic reaction or an arthritic condition), such as a polypeptide that specifically binds TNF-a or IL-6.

[0143] The preferred protein to be secreted by the effector cell is an antibody or fragment thereof, or a polypeptide that is a single-chain single-domain polypeptide, such as a VHH nanobody or scFv protein. One class of proteins that can be secreted are immune checkpoint receptor antagonist or agonist antibodies with or without Fc domains. However, other proteins can be expressed and secreted by the engineered effector cells, such as cytokines or other immunomodulatory proteins. For example, antibodies, antigen-binding portions of antibodies, or single-chain antibodies (e.g., VHH nanobodies) against PDL1, CTLA-4, CD-40, LAG-3, TIM-3, BTLA, CD160, 2B4, CD40, 4-1BB, GITR, OX-40, CD27, HVEM, or LIGHT can be expressed and secreted by the effector cells. Cytokines secreted by the effector cells can include TGF-b, VEGF, TNF-a, CCR5, CCR7, IL-2, IL-7, IL-15, and IL-17, for example. The engineered effector cells disclosed herein can express and secrete two or more different types of effector polypeptides, including different antibodies, cytokines, or combinations. For example, the engineered effector cells can secrete a PDL1 antibody and a CTLA-4 antibody, or a PDL1 and a VEGF antibody.

[0144] An example of a secreted effector protein is an anti-PD-1 VHH antibody having the amino acid sequence: QVQLVESGGGLVQAGGSLRLSCAASGDTSFISAAGWYRQAPGKERELVAAITNTGITYYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCNAGAPPPGGLGYDESDYWGQGTQVTVSS (SEQ ID NO: 3).

[0145] The engineered host immune cells can be various T cells, CIK (cytokine-activated killer cells), DC-CIK (dendritic cells / CIK), NK cells (natural killer cells), NKT cells (natural killer T cells), stem cells, TIL (tumor infiltrating lymphocytes), macrophages, and other immune cells. The host immune cells are typically autologous cells of the subject being treated for the disease.

[0146] In some embodiments, the engineered immune cells are transformed with a vector comprising a coding sequence construct having at least three structural components: a first polynucleotide encoding a first domain comprising an intracellular signaling domain that activates a transcriptional program in an "activated" T cell, such as a CD3 epsilon (CD3e) or CD3 zeta (CD3z) domain of a T cell surface glycoprotein; a second polynucleotide encoding a domain comprising a transmembrane domain (and optionally a spacer peptide), such as a domain of a CD28 protein; and a third polynucleotide encoding a "marker" polypeptide domain that specifically binds to another polypeptide that activates the transcriptional program in the host immune cell (e.g., T cell) through the intracellular signaling domain.

[0147] The intracellular signaling domain can comprise a domain involved in immune co-stimulatory signaling (e.g., a B7 binding domain), and additionally or alternatively, an ITAM domain of CD3e. Preferably, the ITAM domain comprises the amino acid sequence YMNM (SEQ ID NO: 4).

[0148] In some embodiments, the transmembrane domain and the intracellular signaling domain are both domains of a CD28 protein.

[0149] In some embodiments, the signal transduction domain comprises an immune co-stimulatory domain linked to a CD3e domain, such as CD28 / CD3e, 4-1BB / CD3e, ICOS / CD3e, CD27 / CD3e, OX40 / CD3e, or CD40L / CD3e.

[0150] The marker domain polypeptide is preferably a polypeptide that is not expressed or minimally expressed in adult tissues. For example, the marker polypeptide can be derived from a protein that is expressed only or primarily in embryonic human cells (i.e., a "fetal protein"), or the marker polypeptide can be a wholly synthetic amino acid sequence.

[0151] Examples of fetal proteins from which the marker polypeptide can be derived include fetal proteins expressed during embryogenesis, such as Oct-4, Sox-2, and Klf-2. In some embodiments, a portion of the full-length protein is used; for example, polypeptides 20-100 amino acids in length are often used. The amino acid sequences of Oct-4, Sox-2, and Klf-2 are as follows:

[0152] Oct4:

[0153] MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGI (SEQ ID NO: 5)

[0154] Sox-2:

[0155] MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSR (SEQ ID NO: 6)

[0156] Klf-2:

[0157] MALSEPILPSFSTFASPCRERGLQERWPRAEPESGGTDDDLNSVLDFILSMGLD (SEQ ID NO: 7)

[0158] The marker domain portion of the ICAP can be a polypeptide having the amino acid sequence: MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSF (SEQ ID NO: 8).

[0159] The ICAP marker domain can contain a structurally inert domain from the human mesothelin ECD. For polypeptides encoding a mesothelin domain, it contains a peptide sequence of Domain I, II, or III as shown below:

[0160] EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL (Domain I - SEQ ID NO: 9)

[0161] SLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQN (Domain II - SEQ ID NO: 10)

[0162] CSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKL (Domain III - SEQ ID NO: 11)

[0163] A marker polypeptide can be derived from a structural membrane protein that does not have an intracellular signal transduction function or does not interact with other biologically active molecules, to provide a "structurally inert" domain of the structural membrane protein, provided that the structural membrane protein normally associates with another protein or carbohydrate such that the epitope constituting the marker domain is not exposed to an antibody in vivo. The marker polypeptide preferably has little or no immunogenicity. The immunogenicity of a marker polypeptide can be determined by 1) computer algorithmic calculation of the number of T cell epitopes; 2) in vitro assays to determine T cell activation potential; and 3) in vivo experiments using animal models.

[0164] Any of the marker domains described above can be combined with any of the transmembrane domains described above and any of the intracellular signaling domains described above to form an ICAP polypeptide. Short polypeptide linkers can be used to join the domains of the ICAP.

[0165] For example, any of the marker domains described above can be encoded as Figure 3B the "marker domain" portion of plasmid pNB338B-ICAPs-VHH shown below.

[0166] This construct is expressed in an effector cell to produce an "immune cell activation polypeptide" (ICAP) that localizes to the extracellular membrane, such that the marker domain is extracellular.

[0167] The effector cells disclosed herein can be used with bispecific polypeptides - i.e., polypeptides having two functional domains linked by a linking polypeptide or by chemical conjugation, each domain having the activity of specifically binding a different ligand. Herein, in some embodiments, the bispecific polypeptides are also referred to as "VHH-TCPs", as a preferred form of bispecific polypeptides comprising two or more single-chain nanobodies (single-chain, single-domain antibodies).

[0168] One domain of the bispecific polypeptide (L-bd) comprises an amino acid sequence that specifically binds to a marker domain of an ICAP on the surface of an effector cell, and one domain of the bispecific polypeptide (CSP-bd) specifically binds to a protein on the surface of a "target", which is preferably a cellular target, such as a tumor cell, but can be any cell or surface that binds to the target protein. Such surface-presented target polypeptides are referred to herein as "cell surface proteins" or epitopes thereof.

[0169] Such cell surface proteins can be antigens associated with tumors, autoimmune diseases, or cellular or organismal aging, such as CD19, mesothelin, BCMA, EGFR, vimentin, Dcr2, or DPP4. In some embodiments, the target cell is a cell, such as a B cell, mesothelial cell, breast cell, or tumor of fibroblast cells, that abnormally expresses one or more of these proteins, either in quantity or mutant protein.

[0170] The bispecific polypeptide (referred to herein as VHH-TCP) can comprise additional domains to provide additional binding, or biochemical or physiological activity, such as recognizing multiple epitopes from the same target protein or epitopes from multiple target proteins (referred to as "multispecific polypeptides", including, by way of example, trispecific polypeptides, tetraspecific polypeptides, pentaspecific polypeptides, hexaspecific polypeptides). The bispecific polypeptide can also comprise one or more binding motifs to recognize human IgG Fc domains as marker domains of ICAPs to enable effector cell activity switching through ADCC, CDC, and ADCP mechanisms.

[0171] Additionally or alternatively, the bispecific (multispecific) polypeptide (VHH-TCP) can also include one or more domains derived from serum albumin having different molecular weights to control the half-life of the bispecific polypeptide in vivo.

[0172] A domain for binding a fluorophore can be included in the bispecific polypeptide to allow tracking of the bispecific polypeptide and the cells to which the bispecific polypeptide specifically binds in vivo, for example, by examination of fluorescently stained tissue samples.

[0173] Preferably, the domains of the bispecific polypeptide can be connected to each other N-terminus to C-terminus by one or more linker peptides. The length of the linker can be adjusted to adjust the molecular weight of the bispecific polypeptide or the spatial interactions between its domains (e.g., to reduce spatial interactions).

[0174] The linker portion of the bispecific polypeptide can also include an amino acid sequence that is susceptible to cleavage by a peptidase 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) can be cleaved by matrix metalloproteinase-1, while the amino acid sequence VSQTSKLTRAETVFPDV (SEQ ID NO: 15) can be cleaved by Factor IXa / Factor VIIa.

[0175] In some embodiments, one or more (e.g., all) of the active domains comprise a VHH nanobody polypeptide.

[0176] L-bd can be a single antibody domain, a VHH domain derived from a camelid IgG. The CDR3 region of such a VHH domain can comprise 15-20 amino acids that act as a paratope that binds to one or more epitopes on the marker domain.

[0177] The bispecific polypeptide can include an L-bd (a VHH domain that specifically binds to a marker polypeptide) and a CSP-bd (a VHH domain that specifically binds to CD19 or CD20). Such a bispecific polypeptide can be used to treat B-cell lymphoma, e.g., non-Hodgkin lymphoma. In some embodiments, the bispecific polypeptide can include an L-bd (a VHH domain that specifically binds to a marker polypeptide) and a CSP-bd (a VHH domain that specifically binds to EGFR). The amino acid sequence of the CDR3 region from a VHH antibody can bind to EGFR on the surface of non-small cell lung cancer cells. Such a bispecific polypeptide can be used to treat non-small cell lung cancer.

[0178] The bispecific polypeptide can include an L-bd (a VHH domain that specifically binds to a marker polypeptide) and a CSP-bd (a VHH domain that specifically binds to CPC3). In some embodiments, the bispecific polypeptide can include an L-bd (a VHH domain that specifically binds to a marker polypeptide) and a CSP-bd (a VHH domain that specifically binds to BCMA). In some embodiments, the bispecific polypeptide can include an L-bd (a VHH domain that specifically binds to a marker polypeptide) and a CSP-bd (a VHH domain that specifically binds to HER2). Such a bispecific polypeptide can be used to treat HER2 + breast cancer tumor.

[0179] An exemplary bispecific polypeptide comprising two VHH domains linked by a linker (VHH + linker + anti-EGFR VHH bound to a marker domain comprising a structurally inert peptide derived from human mesothelin ECD) linked by a linker, the amino acid sequence of which is as follows: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSS (SEQ ID NO: 16).

[0180] Preferably, the binding of the bispecific polypeptide to epitopes on other cells than the target cells has no significant effect on the pharmacokinetics or pharmacological distribution of the bispecific polypeptide in vivo, and preferably, when such binding occurs, it does not cause any physiologically significant observable effect other than the activation of effector cells expressing the relevant marker domain to be bound by the bispecific polypeptide.

[0181] By means of the embodiments illustrated and described herein, the Applicant has devised a method for treating tumors using the engineered effector cells and bispecific polypeptides disclosed herein, and variants thereof.

[0182] In one such method, the engineered immune cells, which can be T cells or other cell types described herein, are injected directly into a solid tumor as effector cells. Alternatively, the engineered immune cells can be administered intravenously (IV, for example when treating leukemia or lymphoma). Different administration methods can be performed depending on the disease indication. In most cases, intravenous administration is performed to treat the disease. Intraperitoneal administration can be used to treat malignant pleural mesothelioma (MPM).

[0183] For the treatment of solid tumors, direct injection into the tumor is expected to result in better distribution of the cells in the tumor microenvironment (more engineered immune cells in the vicinity of the target tumor cells).

[0184] In a typical treatment method, the VHH-TCP can be administered in an amount of 10 ng / ml to 100 ng / ml, the concentration of engineered immune cells being, for example, 5 x 10 4 , 1 x 10 5 , 5 x 105 or 1 x 10 6 engineered cells / ml.

[0185] In one example embodiment of the method of treatment without VHH-TCP activating molecules, the engineered immune cells are T cells expressing ICAPs with a VHH tag domain that specifically binds to CD19 on B cells and with intracellular signaling domains and transmembrane domains of common T cell receptors (i.e., CD28 and CD3e). The engineered T cells also contain a vector expressing an anti-PD-1-Fc effector polypeptide under the control of a constitutive promoter. After administration of the cells to a subject, the tag VHH domain of the ICAPs specifically binds to CD19 on B cells, which transduces a signal to the engineered immune cells, which are then activated via CD3 and CD28 intracellular signaling and proliferate in the vicinity of the B cell target. The proliferating cells secrete large amounts of anti-PD1 effector proteins in the vicinity of the bound B cells.

[0186] In various embodiments, the disclosed system provides one or more of the following advantages. Not every embodiment will exhibit every advantage.

[0187] In embodiments of ICAP tag domains, polypeptides derived from fetal proteins or structural membrane proteins provide a wide range of possible L-bd for VHH-TCP binding, and because the domains are non-immunogenic or less immunogenic, safety of cell therapy can be improved.

[0188] The diversity of domains that can be included in the bispecific polypeptide (VHH-TCP) provides the ability to alter many characteristics, such as VHH-TCP affinity to effector cells, and the range of cells that can be targeted by the CSP-bd. Additional functional domains can also be added, and the epitope binding valence can be modulated for efficient and safe use of the system to treat disease.

[0189] The bispecific polypeptide (VHH-TCP) that specifically binds to a tag domain of an immune cell activator polypeptide (including a signaling domain that activates immune cell host proliferation) and specifically binds to a cell surface protein of a B cell (such as a CD19 ligand) can induce proliferation of effector cells in vivo, thereby increasing the number of immune effector cells in the vicinity of the B cell binding, which in turn increases the amount of effector polypeptide. This saves time and avoids the cost of producing effector polypeptides in vitro.

[0190] The bispecific polypeptide (VHH-TCP) can be engineered in a variety of forms to optimize the activity of the effector cells by the length and flexibility of the linker between the VHHs in the bispecific polypeptide (VHH-TCP), the position of each binding motif, and the overall size of the VHH-TCP.

[0191] The activity of the effector cells in vivo can be controlled by administering different amounts of the bispecific polypeptide (VHH-TCP) and / or controlling the half-life of the VHH-TCP. This is a novel and comprehensive approach to minimize the toxicity of CAR-based therapies.

[0192] In addition, the effector cells can be activated by ADCC effect using a suitable marker protein that specifically binds to the Fc epitope.

[0193] Importantly, the features of nanobodies (e.g. small size, high stability and ease of engineering) provide unique advantages for optimizing in vivo therapeutic systems.

[0194] Stopping the administration of VHH-TCP to the subject can prevent adverse reactions associated with sustained effector cell activity, while also providing the opportunity for subsequent VHH-TCP administration for disease relapse events.

[0195] The in situ secretion of antibodies (preferably nanobodies secreted by activated effector cells) can inhibit or stimulate immune checkpoint receptors, thereby improving targeting of solid tumors through penetration, proliferation and persistence of the TME (tumor microenvironment). Nanobody bispecific polypeptides have an advantage over conventional antibodies in penetrating the TME due to their small size and stability.

[0196] The effector cell-bispecific polypeptide (VHH-TCP) system disclosed herein can improve many pitfalls of current CAR-T therapies: for example, by targeting multiple tumor antigens with a single standardized immune receptor, and the diverse structure of VHH-TCPs can be used to control and optimize immune cell activity. Treatments utilizing the disclosed system are expected to exhibit less toxicity or side effects. In addition, the components of the system can be easily and low-cost manufactured. The diversity of ligands and binding domains that can be incorporated into ICAPs and bispecific polypeptides (VHH-TCPs) allows the modular system to be used to treat a variety of diseases or conduct research, for example, incorporating a FITC binding domain into VHH-TCPs can be used to track the fate of activated effector cells in vivo.

[0197] EMBODIMENT

[0198] Example 1: Representative working examples

[0199] 1. Generation of modified effector T cells by electroporation

[0200] ICAP includes a marker polypeptide (27 amino acids of fetal protein or mesothelin), a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain (CD28IC), and CD3 zeta. 1182-Fc(EQ) comprises VHH-1182 and an IgG4 Fc domain.

[0201] The 1182-Fc(EQ) structural gene was cloned into the piggyBac transposon vector pS338B to obtain the plasmid pS338B-1182-Fc(EQ) (SEQ ID NO: 1). The ICAP-VHH gene was PCR-amplified and cloned into the piggyBac transposon vector pNB338B to obtain the plasmid pNB338B-ICAP-VHH (SEQ ID NO: 2). The ICAP-VHH gene was replaced with an empty multiple cloning site (MCS) gene to generate the MOCK construct plasmid. Figure 3A Figure 3B Human peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased from AllCells (Shanghai, China). PBMCs were cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) for 0.5-1 hour at 37°C in a 5% CO2 humidified incubator, then harvested and washed twice with Dulbecco’s phosphate-buffered saline (PBS).

[0202] PBMCs were counted according to the manufacturer’s instructions and used to isolate T cells using the Dynal® Human T Cell Isolation Kit.

[0203] PBMCs were counted according to the manufacturer’s instructions and used to isolate T cells using the Dynal® Human T Cell Isolation Kit. Human T cells T cells transfected with ICAP-VHH / 1182-Fc(EQ) plasmid or MOCK / 1182-Fc(EQ) plasmid were then specifically stimulated in 6-well plates coated with anti-CD3 antibody / anti-CD28 antibody (5 μg / mL) for 4-5 days. The transformed T cells were then cultured in AIM-V medium containing 2% FBS and 100 U / mL recombinant human interleukin 2 (IL-2) for 10 days to produce sufficient amounts of effector T cells.

[0204] 2. Transduction efficiency assay

[0205] The transduction efficiency of the marker polypeptide into T cells was determined by flow cytometry using biotin-conjugated anti-IgG4(Fc) antibody and PE-conjugated streptavidin secondary antibody.

[0206] 3. Binding efficiency assay

[0207] The binding of the bispecific polypeptide to ordinary T cells was measured by flow cytometry using anti-CD19-PE antibody. The proportion of cells positive for CD19 and the marker (e.g., meso) was compared to determine the binding efficiency.

[0208] 4. Proliferation capacity assay (incubation with bispecific VHH and tumor cells)

[0209] ​1 x 10 7 carboxyfluorescein succinimidyl ester for 10 minutes and recovered in culture medium for another 10 minutes. 5 x 10 5 cells were counted and co-cultured with tumor cell lines expressing different antigens (including BCMA, EGFR, mesothelin and GPC3) and bispecific VHH for 7 days, replacing the culture medium every 3-4 days with fresh medium (AIM-V + 2% FBS). Proliferation of effector cells was then determined by flow cytometry.

[0210] 5. Quantification of 1182-Fc-VHH secretion

[0211] 5 x 10 5 cells were seeded in 6-well plates with 1 ml of culture medium, then tumor cells and bispecific VHH were added and co-cultured for 48 hours. Effector T cell suspension was then centrifuged at 3000 rpm for 3 minutes; supernatant was retained and 1182-Fc protein was quantified by ELISA.

[0212] 6. Cytotoxicity assay (for adherent cell lines)

[0213] Cytotoxicity of effector T cells transduced with the marker construct or vector control was determined by using the impedance-based xCELLigence RTCA TP instrument.

[0214] Target tumor cells were seeded in a resistor-bottomed 96-well plate in the RTCA TP instrument at 10,000 cells per well overnight (more than 16 hours). Bispecific VHH antibodies were added to the cultured target tumor cells and the cells were further cultured for 30 minutes. Then the transformed T cells carrying plasmids pS338B-1182-Fc(EQ) and pNB338B-ICAP-VHH (effector cells) were incubated with target tumor cells at different effector: target cell ratios for about 100 hours (end point depending on the killing efficiency of the transformed T cells). During the experiment, the cell index values are closely related to tumor cell adhesion, so a lower cell attachment indicates a higher cytotoxicity and is detected by the RTCA system and the ACEA Multi-Label Reader (PerkinElmer). Real-time killing curves were automatically generated by the system software. Specific lysis (%) per transformed T cell was calculated using end-point data [Specific lysis = (cell index of tumor cells alone - cell index of transformed T cells co-cultured with tumor cells) / cell index of tumor cells alone].

[0215] 7. Cytotoxicity assay (for suspension cell lines)

[0216] According to the manufacturer's protocol ( EuTDA Cytotoxic Reagent AD0116 (PerkinElmer) was used to determine the cytotoxicity of effector T cells transduced with labeled constructs or vector controls. In short, target tumor cells were 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) were placed in a V-bottom plate containing a bispecific peptide that specifically binds to both target tumor cells and effector cells (i.e., transformed T cells), with 100 μL of effector cells added at varying cell concentrations. After incubation at room temperature for 15 minutes, 20 μL of the supernatant was transferred to 200 μL of Europium solution. Fluorescence was measured using a time-resolved fluorometer. Specific release (%) = Experimental releases (number of times) - Spontaneous releases (number of times) / Maximum releases (number of times) - Spontaneous releases (number of times) x 100.

[0217] 8. In vivo specific targeting activity

[0218] NOD-SCID IL2 Rγ were fed under pathogen-free conditions. - / - (NSG) mice (Shanghai, China). Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC). To establish a xenograft tumor model, NSG mice were subcutaneously inoculated with an equal volume of Matrigel. TM Mixed 5×10 6 EGFR + Lung tumor cells and 5×10 6 MSLN + Ovarian tumor cells. Tumor dimensions were obtained using calipers, and tumor volume was calculated based on the following formula: V = 1 / 2 (length × width) 2 When the tumor burden is approximately 100 mm 3 At day 5, effector T cells labeled with Flux and a bispecific VHH targeting EGFR were administered intravenously. Specific targeting of effector T cells to the lungs was confirmed by bioluminescence imaging (BLI). On day 5, another bispecific VHH targeting MSLN was administered intravenously to observe specific targeting of effector T cells to ovarian tumor cells. In vivo proliferation of effector T cells was monitored using bioluminescence imaging with a Xenogen IVIS imaging system (PerkinElmer, USA).

[0219] 9. In vivo proliferation and antitumor activity

[0220] To establish a xenograft tumor model, 5×10 6 Fluc-labeled tumor cells with an equal volume of Matrigel TM The mixture was then subcutaneously injected into NSG mice. When the tumor burden was approximately 100 mm... 3At the time, mice were randomly divided into three groups (5 mice per group) and intravenously (iv) injected with MOCK-T, effector T cells or PBS vehicle containing polypeptide VHH, the time point was designated as day 0.

[0221] Peripheral blood was taken from all mice via the tail vein to detect the proliferation of effector T cells and the copy number of ICAP genes. After reaching the moribund state, mice were euthanized and then bone marrow, blood and spleen were collected. The percentage of CD3 + T cells and memory T cell subsets in the spleen were analyzed by flow cytometry. During the entire in vivo experiment, the body weight of mice was measured using an electronic balance. The progression of tumors was confirmed by bioluminescence imaging (BLI) using a Xenogen IVIS imaging system (PerkinElmer, USA). All measurements were performed every five days.

[0222] 10. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC)

[0223] H&E and immunohistochemistry were performed to evaluate the safety of super cell therapy. Mouse tissues (heart, liver, spleen, lung, kidney and brain) were fixed with formalin and then embedded with paraffin. The tissues were serially sectioned into 4 pm thickness using a RM2245 microtome (Leica, Germany) and then stained with H&E. To detect the infiltration ability of effector T cells in tumor tissues, IHC analysis was performed using anti-CD3 antibody (Abeam, #ab16669) at a dilution of 1:100. Images were taken using an AXIOSTAR PLUS microscope (ZEISS, Germany).

[0224] 11. Tissue distribution assay

[0225] Real-time quantitative PCR (RT-qPCR) was used to determine the tissue distribution of 1182-VHH, transfected T cells and adaptor VHH proteins. Mouse tissues (heart, liver, spleen, lung, kidney and brain) were digested to prepare a single cell suspension. Total DNA was extracted from T cells using a genomic DNA extraction kit Ver.5.0 (TAKARA, China) according to the manufacturer's instructions. Real-time polymerase chain reaction was performed using TaqMan TM Universal Master Mix II (ThermoFisher Scientific, USA). Primers and probes for CAR and actin were synthesized or labeled by Generay Biotech Co., Ltd. (Shanghai, China). Real-time quantitative PCR reactions were performed in two steps: (1) pre-incubation: 95°C for 5 minutes; (2) amplification: 40 cycles of (95°C for 20 seconds, 60°C for 1 minute). All reactions were repeated three times.

[0226] 12. Statistical analysis

[0227] All data are presented as mean ± SD. T-test was used to assess differences between two independent groups. One-way ANOVA was used to compare whether there was a statistically significant difference between three or more independent groups. Two-way ANOVA was used to determine the effect of two nominal predictor variables on a continuous outcome variable. All statistical analyses were performed using Graphpad Prism version 7 software (La Jolla, CA). All data with error bars are presented as mean ± SD. The following were considered statistically significant differences: P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

[0228] Example 2: Identification and characterization of a VHH sequence with high affinity to MSLN, BCMA or EGFR

[0229] It is described the use of alpaca immune libraries to identify and characterize a specific VHH nanobody against MSLN, BCMA or EGFR with high affinity.

[0230] 1. VHH nanobody against MSLN

[0231] For the first immunization, 400 pg of MSLN-hFc emulsified with Freund's complete adjuvant was administered subcutaneously to each alpaca. Two weeks later, 200 pg of MSLN-hFc emulsified with Freund's incomplete adjuvant was administered subcutaneously. Thereafter, 5 additional immunizations were performed every other week with 200 pg of MSLN-hFc emulsified with Freund's incomplete adjuvant. ELISA and FACS confirmed high serum titers against MSLN-His antigen as well as the HEK293T-MSLN stable cell line.

[0232] Seven days after the last injection, 50 mL of blood was collected, lymphocytes were purified from the sample, RNA was extracted and used for immune library construction. Two rounds of solid protein panning were performed with the MSLN-His antigen followed by ELISA screening and FACS validation. A positive clone named M2339 (VHH) was obtained.

[0233] The antibody was expressed as a hFc fusion protein, designated M2339 (VHH), using procedures described in patent publication WO2020176815A2 (herein incorporated by reference in its entirety and for all purposes). The binding affinity of M2339 (VHH) to MSLN antigen was tested by surface plasmon resonance (SPR). First, M2339 (VHH) was captured by a sensor chip pre-immobilized with protein A, the antibody was captured by protein A. Then MSLN-His protein was used as the flow phase at five different concentrations, the association time and dissociation time were 30 min and 60 min, respectively. The association rate constant (k on ), dissociation rate constant (k off ) and equilibrium constant (K D ) were analyzed using Biacore evaluation software 2.0 (GE). As shown in Table 1 below, M2339 (VHH) has high affinity to MSLN antigen, K D is 2.64E-10.

[0234] The binding affinity of M2339 (VHH) to HEK293T-MSLN cells was identified by flow cytometry. HEK293T cells and HEK293T-MSLN cells were incubated at 3x10 5 cells / well in different wells of a 96-well plate, followed by incubation with serially diluted M2339 (VHH) for half an hour, incubation with secondary antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008), and then detection with a CytoFLEX flow cytometer. The “isotype” is an isotype control (negative control). As shown in FIG. A, M2339 (VHH) showed good specific binding affinity to HEK293T-MSLN cell line. Figure 4

[0235] 2. VHH Nanobodies against BCMA

[0236] This example describes the identification and characterization of a specific VHH nanobody with high affinity to BCMA using an alpaca immune library. The alpaca immunization procedure, blood collection, library construction, solid panning, ELISA or FACS screening of positive clones, antibody purification, and subsequent antibody characterization by SPR and FACS are described in Example 2 above. A positive clone designated B029 (VHH) was obtained.

[0237] As shown in Table 1, B029 (VHH) has high affinity to BCMA-His antigen, K D is 1.25E-10.​

[0238] As Figure 4 B029 (VHH) showed good specific binding affinity to CHOK1-BCMA cell line as shown in Figure B.

[0239] 3. VHH Nanobodies against EGFR

[0240] This example describes the identification and characterization of a specific VHH nanobody against EGFR with high affinity using an alpaca immune library. The alpaca immunization procedure, blood collection, library construction, solid panning, ELISA or FACS screening of positive clones, antibody purification and subsequent antibody characterization by SPR and FACS are described in Example 2 above. A positive clone named E454 (VHH) was obtained.

[0241] As shown in Table 1, E454 (VHH) has a high affinity to EGFR His antigen with a K D of 1.27E-09.

[0242] As Figure 4 C, E454 (VHH) showed good and specific binding affinity to HEK293T-EGFR cell line.

[0243] Table 1: Binding kinetics of M2339 (VHH)-MSLN, B029 (VHH)-BCMA and E454 (VHH)-EGFR

[0244]

[0245]

[0246] Example 3. High affinity VHH specific to mesothelin region II+III

[0247] This example describes the identification and characterization of a specific VHH nanobody against EGFR with high affinity using an alpaca immune library. The alpaca immunization procedure, blood collection, library construction, solid panning, ELISA or FACS screening of positive clones, antibody purification and subsequent antibody characterization by SPR and FACS are described in Example 2 above. A positive clone named E454 (VHH) was obtained.

[0248] Different mesothelin ECD domains with human Fc were expressed in 293T cells and purified by protein A column. Affinity was determined by SPR. Different antigens were captured using a protein A chip, M2339 (VHH) was injected at different concentrations at a flow rate of 10 μl / min, with an association time of 120-180 seconds and a dissociation time of 180-1200 seconds. Binding kinetics were determined in a 1:1 fitting model using Biacore Evaluation software.

[0249] As Figure 5As shown, M2339(VHH) binds to full-length mesothelin, mesothelin I, and mesothelin II+III with different affinities. The mesothelin II+III domain is well recognized by M2339, with a KD value of 4.32E-11M, which is similar to the affinity of the intact mesothelin peptide (Table 2).

[0250] Table 2: Binding kinetics of 2339(VHH) to mesothelin domain

[0251]

[0252] These results indicate that M2339 has a high affinity for mesothelin II+III and can be used as an adaptor VHH, while mesothelin II+III can be used for ICAP of T cells.

[0253] Example 4: Preparation and screening of mesothelin II+III (M-ICAP) based immune cell activating polypeptides

[0254] like Figure 6 As shown, To generate the described M-ICAP-T Different carriers were constructed. For activating immune cells, For example, T cells. All vectors encode the same intracellular regions, including the 4-1BB and CD3ζ intracellular regions. The extracellular regions encoding the polypeptides differ. M-ICAP does not contain any His tag. M-ICAP-his-1 or 2 contain a 6x His-tag at the N-terminus or C-terminus of M-ICAP, respectively. In addition to the mesothelin signal peptide (SP-MSLN), two other signal peptides (SPs) were selected from the 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).

[0255] All vectors were transfected into 293T cells with Lipofectamin 2000 (ThermoFisher, USA) and the expression rate was measured 2-4 days later using flow cytometry. Figure 7 For flow cytometry analysis, blank cell controls were prepared using 19R73-CD19CAR and GFP vectors. M2339-hFc and biotin-conjugated anti-His mAb were used as primary antibodies, and fluorophore-conjugated anti-human Fc and fluorophore-conjugated streptavidin were used as secondary antibodies. (See Table 3 and...) Plasmid name As shown, when the N-terminal His tag of M2339-hFc was detected, the His tag affected the position of the marker (M-ICAP) expression rate, resulting in a higher M-ICAP expression rate. The signal peptide had almost no effect on the M-ICAP expression of M2339-hFc and the anti-His assay.

[0256] Table 3: Expression levels of various M-ICAP peptides

[0257] Frequency (%) Antibody detected Control M2339-IgG1 0.03 GFP pNB338B-19R73 95.44 / Anti-CD19-IgG1 27.56 pNB338B-M-ICAP M2339-IgG1 13.51 pNB338B-SP3-M-ICAP M2339-IgG1 18.66 pNB338B-SP5-M-ICAP M2339-IgG1 17.75 pNB338B-His-1-M-ICAP M2339-IgG1 17.31 pNB338B-His-1-M-ICAP Biotin anti-His mAb 8.97 pNB338B-His-2-M-ICAP M2339-IgG1 9.72 pNB338B-His-2-M-ICAP Biotin anti-His mAb 8 pNB338B-SP3-His-M-ICAP M2339-IgG1 21.19 pNB338B-SP3-His-M-ICAP Biotin anti-His mAb 9.5 pNB338B-SP5-His-M-ICAP M2339-IgG1 18.38 pNB338B-SP5-His-M-ICAP Biotin anti-His mAb 10.56 Figure 8

[0258] Example 5: M-ICAP-T cell construction

[0259] As shown in Figure 8 A, M-ICAP expression vectors containing different signal peptides (SP-MSLN, SP3, SP5) were constructed and fused with T cell activation / signal transduction domains (CD28 / 4-1BB, CD3 zeta) of traditional CAR vectors. The ICAP vector consists of a marker polypeptide M-ICAP (from mesothelin II+III domain), CD28 transmembrane domain, CD28 / 4-1BB intracellular costimulatory signal domain (CD28 / 4-1BB IC), and CD3 zeta domain. The ICAP-VHH gene was amplified by PCR and cloned into the piggyBac transposon vector pNB338B to obtain the plasmid pNB338B-ICAP (SP-MSLN, SP3, SP5) (B). Figure 8 B).

[0260] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased from AllCells (Shanghai, China). PBMCs were cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) for 0.5-1 hour at 37°C in a 5% CO2 humidified incubator, then the cells were harvested and washed twice with Dulbecco’s phosphate-buffered saline (PBS). According to the manufacturer’s instructions, the Human T Cell Kit was used to count and electroporate PBMCs. After that, the transfected T cells were specifically stimulated in 6-well plates coated with anti-His / M2339 (VHH-Fc) and anti-CD28 antibody (5 pg / mL) for 4-5 days, and then cultured in AIM-V medium containing 2% FBS and 100 U / mL recombinant human interleukin 2 (IL-2) for 10 days to produce a sufficient number of effector T cells. Flow cytometry, biotin-conjugated anti-His antibody, and PE-conjugated streptavidin secondary antibody were used to determine the transduction efficiency of the marker polypeptide (M-ICAP expression) on T cells.

[0261] As shown in Example 6: Generation and validation of M-ICAP-T cells C and 8D, the positive rate of M-ICAP-T after amplification was higher than 30% on the 8th day and 92% on the 13th day. All three different ICAPs were activated and amplified by stimulation of M2339 VHH or anti-His antibody. The expression of M-ICAP ECD outside the T cell membrane and the construction of M-ICAP-T cells were successfully achieved.

[0262] Figure 9

[0263] M-ICAP was fused into several different CAR sequences, and M-ICAP-T cells were obtained by electroporation combined with specific activation of donor-derived PBMC cells. The ICAP vector includes a marker polypeptide (from mesothelin II+III domain), a CD28 transmembrane domain, a CD28 / 4-1BB intracellular co-stimulatory signaling domain (CD28 / 4-1BB IC), and a CD3 zeta domain. 1182-Fc(EQ) contains VHH-1182 and IgG4 Fc domain.

[0264] The production of ICAP CAR-expressing cells (ICAP-T cells) or typical CAR-T cells by electroporation is described in Example 5.

[0265] A series of tests were performed after expansion to verify the modified T cells, including ICAP expression rate, expansion effect, CD4 / CD8 positive cell ratio in CD3 positive cells, and effector memory T cell (Tem) / central memory T cell (Tcm) ratio in memory T cell (Tm). The expression rate of the marker polypeptide on the surface of T cells (M-ICAP expression) was determined by flow cytometry using biotin-conjugated anti-His antibody and PE-conjugated streptavidin secondary antibody. As shown in Example 7: Design and characterization of M2339 VHH based TCP As shown, the ICAP-T cells derived from PBMC of two donors (AC1909A and SL2007A) were expanded by up to 10 times during the preparation process, and the ICAP expression rate was as high as 80% (different for different donors); the CD4 / CD8 positive value varied for different donors, and the central memory T cells accounted for the majority of memory cells. Different CAR-element sequences had certain effects on the positive rate and expansion of ICAP-T cells, for example, the proliferation and ICAP expression of M-ICAP-28BB-T cells were less than those of M-ICAP and M-ICAP-28. In terms of specific activation of T cells, we compared the effects of different antibodies / TCPs on the expansion of M-ICAP-transfected PBMCs. As shown, M2339, anti-His antibody, and TCP001-C / P could specifically activate the expansion of ICAP-T cells.

[0266] Figure 10

[0267] This example describes the design and properties of TCP used herein. The TCP used herein is a bispecific antibody that can recognize both the target B cells or tumor-specific antigens (such as CD19, BCMA, and EGFR) of M-ICAP-T cells and the M-ICAP polypeptide (from mesothelin), and thus can be used as an adaptor to control the proliferation or cytotoxicity of M-ICAP-T cells. The TCPs designed and applied in the examples are listed in Table 4.

[0268] Table 4: Domains, molecular weights and purity of TCPs used in the examples

[0269]

[0270]

[0271] 1. Design and purification of TCPs

[0272] BCMA-TCPs designed to simultaneously target BCMA antigen and M-ICAP polypeptide (a marker derived from mesothelin) were designed for cytotoxicity and in vitro efficacy assays described further below. To investigate the effect of different linker formats on the biological activity and stability of TCPs, three formats of BCMA-TCPs (TCP001-C, TCP002-C and TCP003-C) were designed with different linkers (3x GGGGS linker, hlgG4-Fc and hlgG4-CH3, respectively). Meanwhile, MC001C and MC001D were constructed against BCMA and M-ICAP, respectively, as two positive controls in mAb format.

[0273] CD19-TCP with 3x G4S linker to simultaneously target CD19 antigen and M-ICAP polypeptide was named TCP011-P and designed for M-ICAP-T proliferation assay stimulated by CD19 antigen.

[0274] EGFR-TCP with 3x G4S linker to simultaneously target EGFR antigen and M-ICAP polypeptide was named TCP021-P and designed for M-ICAP-T cytotoxicity assay using EGFR-expressing solid tumor cell line as target.

[0275] The N-terminal M2339VHH sequence targeting M-ICAP polypeptide was identified from phage display of llama immunized VHH library as described in Example 2 and Example 3 above. The B029 (VHH) sequence targeting BMCA was identified from phage display of llama immunized VHH library as described in Example 2 above. The scFc sequence in TCP001-P targeting BMCA was derived from B2121 in CN201580050638. The VHH sequence in TCP001-N targeting GFP was derived from GFP-specific VHH described in Kubala et al (M.H. Kubala et al, Protein Sci. 19 :2389-2401 (2010) (hereby incorporated by reference in its entirety to describe such VHHs and ways of using them).

[0276] The scFv sequence in TCP011-P targeting CD19 was derived from FMC063, described in Chinese patent application CN201480027401.4 (herein incorporated by reference in its entirety). The E454 sequence in TCP021-P targeting EGFR was identified from phage display of a llama immune VHH library, as described above in Example 2.

[0277] The genes were synthesized and cloned by Genewiz, Inc. All ORF DNA were cloned into pcDNA3.4 vector between BamHI and EcoRI sites. Antibody expression, purification, and purity quality control described herein were performed according to WO2020176815A2 (herein incorporated by reference to describe such methods).

[0278] 2. Affinity characterization of TCPs

[0279] First, the binding affinity of purified BCMA-TCPs to the BMCA antigen was assessed by SPR. The BCMA-his antigen was coupled to a CM5 chip (GE Healthcare Life Sciences) and then flowed over various anti-BCMA BsAbs at a flow rate of 10 uL / min with a dissociation time of 900 s. The binding kinetics were determined using a 1:1 fitting model. The data suggest that the linker structure can affect the binding affinity, as TCP001-C with a 3xG4S linker has a higher binding affinity than TCP002-C and TCP0031-C with larger linkers (Table 5).

[0280] Table 5: Kinetics of BCMA-TCP binding to BMCA

[0281]

[0282] The binding bioactivity of mesothelin and BCMA overexpressing cells was assessed by flow cytometry. The stable cell lines were harvested using 0.25% trypsin.

[0283] Approximately 5E5 cells were collected per sample and the cells were resuspended in 100 μL / well of the His-tagged test antibody. The cells were then incubated with an anti-His-tag antibody (Genscript, China) and streptavidin-PE (Biolegend, China). The incubation step for each step was performed at 4°C for 1 hour in the dark, and then the cells were washed with 200 μL PBS buffer for 2 times. The washed cells were resuspended in 200 μL PBS buffer and the samples were analyzed by FACS. As shown, TCP002-C has the strongest binding to both cell lines, while the binding strength of TCP003-C is slightly higher than TCP001-C. Figure 11 ​

[0284] 3. Stability of BCMA-TCP in human plasma in vitro

[0285] TCP001C, TCP002C and TCP003C were incubated in 100% human plasma at 37°C for up to 21 days and samples were collected at day 0, 1, 3, 7, 14 and 21, respectively. 96-well plates were coated with mesothelin antigen and after plate blocking and washing, the collected samples were incubated with serially diluted standard samples together with the plates for 1 hour at 37°C. Anti-VHH cocktail-HRP (GenScript, A02016) was used as detection antibody. Absorbance was read at 450 nm. Finally, the measured samples were analyzed according to the fitted curve of the standard sample set.

[0286] As shown in Figure 12 , TCP001-C, TCP002-C and TCP003-C were stable in human plasma in vitro at 37°C for more than 21 days.

[0287] The same flow cytometry procedure was applied for BCMA-TCP to confirm the binding affinity of TCP011-P to CD19 and MSLN overexpressing cells Figure 13 and TCP021-P to EGFR and MSLN overexpressing cells In vitro expansion of M-ICAP-T and TCP on target cells .

[0288] Example 8: Figure 14

[0289] To verify the rapid activation and expansion of ICAP-T cells by TCP and target cell culture, 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 -N, respectively. CD19 positive Daudi lymphoma cells were treated with or without 50 ug / ml of mitomycin C for 2 hours. PBMC-T cells transfected with M-ICAP cells were counted as 5x10 5 and co-cultured with 5x10 5 Daudi cells and TCP011-P or -N for 4 days. Then the proliferation of effector or target cells was analyzed by flow cytometry.

[0290] As shown in Example 9: Dose dependent cytotoxic effect of M-ICAP-T on RPMI-8226 cells , M-ICAP-T cells transfected for 5, 8, 13 days could all effectively expand under the stimulation of Daudi cells in the presence of TCP011-P, with the highest expansion at 5 days and 8 days post-transfection. In addition, activated M-ICAP-T cells could kill Daudi cells.

[0291] Figure 16

[0292] For ICAP-t cells to act on BCMA-positive tumor cells, a TCP that specifically binds to BCMA is required. The TCP001-C and -P ends can simultaneously and specifically bind to both ICAP-T and BCMA cells. To verify that ICAP-T cells combined with a specific TCP can act on BCMA-positive tumor cells and exhibit specific cell lysis / killing effects, we compared the cell lysis / killing effects of ICAP-T or CAR-T cells co-cultured with RPMI-8226 cells or L363 cells (three different E:T ratios) in the presence of different TCPs.

[0293] According to the manufacturer's plan ( EuTDA Cytotoxicity Reagents AD0116–PerkinElmer was used to assess the cytotoxicity of T cells in suspension cell lines. 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-plate containing a bispecific peptide that specifically binds to both target tumor cells and effector cells (i.e., transformed T cells), along with 50 μL of effector cells at different cell concentrations (E:T = 16 / 8 / 4:1). After 3.5 hours of incubation, 10 μL of the supernatant was transferred to 100 μL of europium solution. Fluorescence was measured using a time-resolved fluorometer after 15 minutes of incubation at room temperature. Specific release (%) = Experimental release (counts) - Spontaneous release (counts) / Maximum release (counts) - Spontaneous release (counts) x 100.

[0294] IFNγ secretion from T cells was also measured. IFNγ detection was performed according to the manufacturer's protocol (IFNγ Detection Kit, VAL104–Novus). In short, fresh wash buffer, staining agent, diluent, and standards were prepared according to the instructions. Different concentrations of standards and diluted experimental samples were added to the corresponding wells, 100 μL per well. The reaction wells were sealed with sealing tape and incubated at room temperature for 2 hours. After washing four times with wash buffer, 200 μL of enzyme-labeled antibody was added to each well, and incubated at room temperature for 2 hours. After repeating the washing procedure, 200 μL of pre-mixed chromogenic reagent was added to each well, and the reaction mixture was incubated in the dark for 10–30 minutes. The solution color changed from blue to yellow by adding 50 μL / well of stop solution. OD values ​​were recorded over 20 minutes using a spectrophotometer and analyzed in Excel using the selected "four-parameter equation" to obtain a standard curve using the standard sample group.

[0295] like Example 10: Cytotoxicity comparison of ICAP-T with different TCP combinations on RPMI-8226 / L363 cells and IFNyAs shown, the M-ICAP-T cells combined with TCP001-C exhibit strong specific killing activity against tumor target cells, while the non-specific killing activity against T cells is more pronounced at an E:T ratio of 16:1. When the TCP concentration is >0.025 ug / ml, the cell lysis effect increases at E:T ratios of 8:1 and 4:1. The killing effect is best at TCP001-C concentrations of 0.1 and 0.5 ug / ml at E:T ratios of 16:1 and 8:1. When the TCP001-C concentration reaches 2 ug / ml, the effects of E:T ratios of 16:1 and 8:1 decrease, with E:T ratio of 16:1 showing the best killing effect. At different E:T ratios, the EC50 values ​​of TCP are similar (EC50 = 0.028 (E:T = 16:1), 0.024 (E:T = 8:1), 0.022 (E:T = 4:1)), but the maximum value corresponds to the E:T ratio.

[0296] ​ Secretion detection

[0297] TCP001-C / P, TCP002-C / P, and TCP003-C / P simultaneously bind to both M-ICAP and BCMA. To verify and compare the specific cell lysis effect of ICAP-T cells combined with these TCPs on BCMA-positive tumor cells, cell lysis / killing assays were performed on ICAP-T or CAR-T cells (E:T = 8:1) co-cultured with RPMI-8226 or l363 cells in the presence of various TCPs. Cytotoxicity and IFNγ secretion assays for T cells used in suspension cell lines are described in Example 9.

[0298] like Figure 16 As shown, the combination of M-ICAP-T and TCP001-C exhibited strong specific killing activity against tumor target cells, while TCP001-C (binding only to BCMA-positive cells) or TCP-MD (not binding to BCMA-positive cells) failed to effectively kill FaDu / SK-OV3 cells. Furthermore, the IFNγ secretion data were consistent with the cytotoxicity data. Compared to the negative control group (TCP001-N, TCP-MD, or IgG), the combination of M-ICAP-T and 0.2 μg / ml TCP001-C / P specifically induced IFNγ release in RPMI-8226 and L363 cell lines. The order of IFNγ release was: TCP001 > TCP003 > TCP002.

[0299] Example 11: Cell lysis of FaDu / SK-OV3 cells by ICAP-T cells in combination with TCPs (binding EGFR) Figure 17

[0300] To enable ICAP-T cells to act on EGFR-positive tumor cells, a TCP that specifically binds to EGFR is needed. TCP021-P can bind to ICAP and EGFR at both ends, respectively. To verify the killing effect of ICAP-T cells in combination with the specific TCP on EGFR-positive tumor cells such as FaDu (human pharyngeal squamous cell carcinoma) and SK-OV3 (human ovarian cancer cells), we compared the killing effect of ICAP-T or CAR-T cells co-cultured with FaDu / SK-OV3 cells in the presence of various TCPs.

[0301] Impedance-based RTCA TP instrument and method (xCELLigence) were used for T cell cytotoxicity assay on adherent cell lines. Target tumor cells were seeded in a 96-well plate with a resistor at the bottom in the RTCA TP instrument at 10,000 cells per well overnight (more than 16 hours). Bispecific TCP or antibody was added to the cultured target tumor cells, and the cells were further cultured for 30 minutes. Then ICAP-T or CAR-T cells were incubated with target tumor cells at different effector: target cell ratios for about 100 hours (the endpoint depends on the killing efficiency of the transformed T cells). During the experiment, the cell index value is closely related to the tumor cell adhesion, the lower the cell adhesion, the higher the cytotoxicity, and the RTCA system collects every 5-10 minutes. Real-time killing curves are automatically generated by the system software. The specific killing (Specific lysis) of each transformed T cell (%) was also calculated using the data at the 48h point [Specific killing = (cell index of tumor cells alone - cell index of transformed T cells co-cultured with tumor cells) / cell index of tumor cells alone].

[0302] As shown in Example 12: IFN-g release and cell lysis of Daudi cells by ICAP-T cells with TCPs , similar to EGFR CAR-T, M-ICAP-T in combination with TCP021-P has strong specific killing effect on two different tumor target cells (non-specific killing of T cells is more obvious for E:T = 4:1), while the combination of TCP001-C (only binds to BCMA positive cells) or TCP-MD (does not bind to EGFR positive cells) cannot effectively kill FaDu or SK-OV3 cells.

[0303] Figure 18

[0304] To enable ICAP-T cells to act on B cells, a TCP capable of specifically binding CD19 is necessary. TCP011-P can bind ICAP and CD19 at its two ends, respectively. To verify the effect of ICAP-T cells on CD19-positive B cells after the combination of such specific TCP, we compared the cell lysis and IFN-γ release of Daudi cells when ICAP-T or CAR-T cells were co-cultured with Daudi cells in the presence of various TCPs. The cytotoxicity and IFNγ secretion detection assay for T cells of suspension cell lines is described in Example 9.

[0305] As shown in Example 13: Generation and characterization of VHH-secreting ICAP-T cells , IFN-γ secretion showed significant differences. The combination of M-ICAP-T and TCP011-P was similar to CD19 CAR-T in killing and IFN-γ secretion. Although TCP001-C (binding to BCMA) killed Daudi cells, the level of IFN-γ secreted by T cells decreased significantly. In addition, when combined with TCP-MD (unable to bind Daudi cells), it was unable to effectively secrete IFN-γ.

[0306] Figure 19

[0307] Due to the complexity of the tumor microenvironment, the clinical efficacy of most CAR-T therapies for solid tumors in current clinical trials is low. To enhance the anti-tumor effect of the ICAP-T cell system, M-ICAP-T cells secreting immune checkpoint inhibitors (such as anti-PD-1, an antagonist that inhibits cytokines in tumors, such as anti-TGFβ, etc.) were prepared, which were produced by transfecting human naive T cells with M-ICAP peptide (from MII+III peptide) and plasmid encoding secreted immune checkpoint inhibitors at the same time.

[0308] ICAP expression was detected using FACS 13 days after the preparation of M-ICAP-T. The results are shown in Figure 19 . Compared with the M-ICAP-T control, the nature of the protein secreted as VHH or scFv had no obvious effect on ICAP expression.

[0309] ELISA was used to test the concentration of secreted proteins. The supernatant was added to the antigen-coated 96-well plate, and HRP-conjugated anti-VHH and HRP-anti-His were used for anti-PD-1 VHH, anti-PD-L1 VHH and anti-TGFβ scFv detection, respectively. The results are shown in Example 14: Anti-PD-1 VHH-secreting ICAP-T cells block surface expressed PD-1 As shown in

[0310] Figure 20

[0311] The binding ability of secreted anti-PD-1 VHH was indirectly tested by FACS on T cells expressing PD-1 on the surface. Commercially available anti-PD-1 antibodies were used to check the level of PD-1 expression on T cells in a competition assay. As shown in Example 15: Anti-TGFp scFv secreted by M-ICAP-T cells blocks TGFp-1 induced luciferase cell signaling

[0312] Figure 21 Example 16: In vivo efficacy study of M-ICAP-T cells with TCP001-C in L363-PDL1-LUC orthotopic tumor model

[0313] A commercially available TGFβRII-293T-Luc cell line was used to determine the blocking activity of the secreted anti-TGFβ scFv obtained in Example 13. 5000 TGFβRII-293 cells were seeded and incubated overnight, test samples were added, followed by 5nM TGFβ, after 6 hours the bioluminescence was read with ONE-GLO. The results are shown in Figure 22

[0314] Figure 23 Figure 24

[0315] In situ tumor model experiments are described below, using NPSG mice (NOD- Prkdc scid IL2rg tm1 / Pnk).

[0316] 1. Tumor inoculation, grouping, dosing and animal observation

[0317] (a) L363-PDL1-luc tumor cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin at 37°C in a 5% CO2 atmosphere. Cells growing in exponential phase were harvested and counted for tumor inoculation. Cell number at the time of inoculation was 4.09E+8; viability was 83.65%.

[0318] (b) For efficacy studies, each mouse was intravenously inoculated with 2*10 6 ​​L363-PDL1-luc cells. The tumor inoculation date was defined as day 0. When the tumor volume reached about 9.4E5 on day 8, 24 mice were selected and randomly divided into 7 groups according to the animal body weight and tumor volume. There were 3-5 tumor-bearing mice in each group. According to the category, dose and frequency of administration, seven groups were set. Group 1 was the negative control group, which was only injected with PBS during the whole test phase. Group 2 was another negative control group, which was intravenously injected with high dose (20*E6) anti-PD-1M-ICAP-T cells on day 8, and then subcutaneously injected with PBS 7 times every 2 days. Group 3 was the positive control group, which was intravenously injected with 5*E6 typical BCMA CAR-T (B2121) on day 8, and then subcutaneously injected with PBS 7 times every 2 days. Group 4 and group 5 were two experimental groups, which were injected with low dose (5*E6) and high dose (20*E6) anti-PD-1M-ICAP-T cells, respectively, and then subcutaneously injected with 5mg / kg TCP001-C 7 times every 2 days. Group 6 and group 7 were two other experimental groups, which were injected with 5*E6 and 20*E6 M-ICAP-T cells, respectively, and then subcutaneously injected with 5mg / kg TCP001-C 7 times every 2 days.

[0319] (c) All procedures related to animal handling, care and treatment in this study were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Bomen under the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, accreditation number 001516). During routine monitoring, the animals were checked for any adverse effects of tumor growth and / or treatment on normal behavior, such as effects on activity, food and water consumption (only by observation) and body weight gain / loss (body weight was measured twice a week before dosing and once a day during the dosing phase), eye / hair gloss and any other abnormal effects, including tumor ulceration. When any animal lost 10% of its body weight, the sponsor was notified.

[0320] 2. Body weight; tumor measurement

[0321] Body weight and bioluminescence signals were measured twice a week. The results of body weight changes in tumor-bearing mice are shown in Example 17: Identification and characterization of exemplary BCMA peptide motifs as universal tag system for expansion of CAR-T in vitro and in vivo No abnormal body weight changes were observed in any group during the test period.

[0322] From the 4th day after cell injection, the bioluminescence signals in mice were measured twice a week using IVIS lumina XR, and throughout the study. The signals were quantified by Living Image software. As AntibodyAs shown, during the study period (days 8–26), there was no significant difference in efficacy between group 2 (M-ICAP-T injection only after tumor inoculation) and group 1 (tumor inoculation only). Compared with the two negative control groups (groups 1 and 2), all experimental groups (groups 4, 5, 6, and 7) injected with M-ICAP-T activated by periodically TCP001-C showed significant efficacy against L363-PDL1 in the orthotopic tumor model. Furthermore, the experimental groups (groups 4, 5, 6, and 7) showed similar efficacy to typical BCMA CAR-T therapy (group 3).

[0323] In the established L363-PDL1-LUC orthotopic tumor model, injection of M-ICAP-T cells every two days and regular TCP001-C injections significantly inhibited the tumor.

[0324] 3. Analysis of whole blood anti-PD-1 and TCP001-C concentrations in mice

[0325] Peripheral blood (100 μl) was collected weekly for counting anti-PD-1 and TCP001-C concentrations in mouse whole blood. PD-1 protein (1 μg / ml) was coated overnight in 96-well plates for ELISA binding assay. Diluted samples were added to wells along with diluted standard samples (8 dilutions of 2 ng / ml) and incubated at 37°C for 1 hour. Anti-VHH-mixture antibody was then added as the detection antibody, followed by the assay reagent. Absorbance was read at 450 nm. Concentrations were determined using a standard curve analysis, as in Example 9.

[0326] like Kon(1 / Ms) As shown, anti-PD-1 VHH was only significantly detected at two sampling time points (day 15 and day 22) in groups 2, 4, and 5. Anti-PD-1 M-ICAP-T cells were used as effector T cells in groups 2, 4, and 5, indicating that anti-PD-1 M-ICAP-T cells can successfully secrete PD-1 in vivo.

[0327] The analytical method for TCP001-C concentration has been described in the "Stability Assessment of Antibodies in Human Plasma" section of the antibody production and characterization section (Example 5). High concentrations of TCP001-C were detected in peripheral blood collected 24 and 48 hours after subcutaneous injection. This indicates that the half-life of TCP001-C in vivo exceeds 48 hours, and the frequency of injection every two days is sufficient to support the efficacy of M-ICAP-T against L363 tumor cells.

[0328] Koff(1 / s) KD(M)

[0329] Peptide motif (about 20-30 amino acids long) fused to the N-terminal of antigen binding domain of CAR-T cell receptor (scFv or VHH) as universal ICAP for CAR-T expansion in vitro or in vivo. The following criteria are used for peptide motif design.

[0330] First, the length of the peptide is about 20-30 amino acids. Second, a nanobody specific to this peptide motif with high binding affinity (KD<1 nM) can be obtained. Finally, when the peptide is fused to the N-terminal of the antigen binding domain of the chimeric antigen receptor of CAR-T cells (scFv or VHH), the nanobody targeting the peptide successfully induces CAR-T expansion in vitro or in vivo.

[0331] 1. Identification and characterization of a VHH sequence with high affinity to MSLN

[0332] A VHH nanobody with high affinity to MSLN was identified using a llama immunization library. The llama immunization screening procedure, blood collection, library construction, solid panning, ELISA or FACS screening of positive clones, antibody purification, and subsequent antibody characterization by SPR and FACS are described in Example 2. One positive clone named anti-MSLN-1444 VHH was obtained.

[0333] As shown in Table 6, anti-MSLN-1444 has a high affinity to the VHH His antigen of MSLN, with a KD of 2.10E-09.

[0334] Table 6: Kinetics of BCMA-TCP binding to BMCA

[0335] 1444 (VHH) Figure 25 Figure 26 Figure 27 Analyte Ligand 5.37E+04 7.50E-05 2.10E-09

[0336] As shown in Table 6, anti-MSLN-1444 has a high affinity to the VHH His antigen of MSLN, with a KD of 2.10E-09. ka(1 / Ms)

[0337] 2. Identification of BCMA peptides (BCMA ICAP) that can be effectively recognized by BCMA full-length VHH binders

[0338] BCMA peptide motifs (~20 aa) can be recognized by some BCMA candidate binding proteins from the previously prepared immunization library with BCMA-hFc antigen with high affinity, and a number of candidate VHH sequences were designed, which have various binding properties to full-length BCMA. BCMA mut1 from native BCMA (1-23 aa of BCMA ECD domain, Table 7— SEQ ID NO: 17) was selected, and the fusion polypeptide of the anti-MSLN-1444 VHH sequence targeting MSLN to be expressed and BCMA mut1 is as follows:​kd(1 / s) Figure 1 shows the sequence of BCMA mutl (SEQ ID NO: 18).

[0339] We then filtered out three VHH sequences with high affinity to it (described below #36, #102 and #367). The sequence of BCMA mutl is also shown in Table 7 (SEQ ID NO: 18), the three VHH sequences were expressed as hFc fusion proteins, named 36(VHH), 102(VHH) and 367(VHH), the process is described in patent publication WO2020176815A2 (herein incorporated by reference in its entirety and for all purposes). The binding affinity of the three VHH nanobodies was measured by SPR. As shown in KD(M) Figure 2, the three VHHs showed high affinity binding to BCMA mutl. The binding kinetic parameters of the three VHHs are shown in Table 8. Anti-BCMA VHH 36# was chosen as the stimulator because it has a higher affinity for BCMA ICAP BMCA mutl.

[0340] Table 7: Sequence of BCMA ICAP BCMA mutl

[0341]

[0342] Table 8: Kinetic parameters of anti-BCMA VHH to BCMA ICAP BCMA mutl

[0343] BCMA mut1#36 BCMA mut1#367 BCMA mut1#102 Figure 28 Figure 28 4.33E+07 1.24E-03 2.87E-11 Figure 29 6.55E+06 8.41E-04 1.29E-10 Figure 30 6.99E+06 0.001547 2.22E-10

[0344] 3. BCMA ICAP can be used to specifically expand CAR T cells with BCMA ICAP

[0345] As shown in Figure 30 Figure 3A, the ICAP-1-23-3GS vector was constructed, in which BCMA mutl (ICAP) was connected with a (G4S)3 linker at the N-terminus of the anti-MSLN CAR. BCMA mutl- MSLN-1444 CAR-T cells were prepared by the following process: transfection with BCMA mutl- MSLN-1444 vector, then stimulation with coated MSLN and anti-CD28 or anti-BCMA mutl 36# and anti-CD28, respectively. The above-mentioned CAR-T cells showed comparable expansion capacity in 2 donors (Figure 3B, 28C and ​ Figure 3B, 28C and ​ Figure 3B, 28C and

[0346] 4. Anti-BCMA mutl 36# does not stimulate non-specific expansion of CAR T cells of BCMA ICAP

[0347] To test the specificity of anti-BCMAmut136# stimulated BCMA ICAP CAR-T cells, as ​ A. MSLN-1444 CAR vectors were constructed and made by transfecting PBMC and then stimulating with MSLN and anti-CD28 or anti-BCMAmut136# and anti-CD28, respectively. Results are shown in ​ B. Only those CAR T cells stimulated with MSLN and anti-CD28 showed clear expansion. MSLN-1444 CAR stimulated with anti-BCMA36# and anti-CD28 did not show expansion in 2 donors.

[0348] Other nucleic acid and amino acid sequences

[0349] MSLN Region II + Region III (M3) DNA:

[0350] TCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCAACAAAGGGCACGAAATGAGTCCTCAGGTGGCCACCCTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACACCCTGACCGCCTTCTACCCTGGGTACCTGTGCTCCCTCAGCCCCGAGGAGCTGAGCTCCGTGCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTGTGACCCAAGGCAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGTCCGAATACTTCGTGAAGATCCAGTCCTTCCTGGGTGGGGCCCCCACGGAGGATTTGAAGGCGCTCAGTCAGCAGAATGTGAGCATGGACTTGGCCACGTTCATGAAGCTGCGGACGGATGCGGTGCTGCCGTTGACTGTGGCTGAGGTGCAGAAACTTCTGGGACCCCACGTGGAGGGCCTGAAGGCGGAGGAGCGGCACCGCCCGGTGCGGGACTGGATCCTACGGCAGCGGCAGGACGACCTGGACACGCTGGGGCTGGGGCTACAGGGCGGCATCCCCAACGGCTACCTGGTCCTAGACCTCAGCATGCAAGAGGCCCTCTCG (SEQ ID NO: 19)

[0351] MSLN Region II + Region III (M3) protein:

[0352] SLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALS (SEQ ID NO: 20)

[0353] M-ICAP CAR ORF DNA:

[0354]

[0355] M-ICAP CAR ORF protein:

[0356] MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22)

[0357] M-ICAP-SP3 CAR ORF DNA:

[0358]

[0359] M-ICAP-SP3 CAR ORF protein:

[0360] MKHLWFFLLLVAAPRWVLSHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24)

[0361] M-ICAP-SP5 CAR ORF DNA:

[0362]

[0363] M-ICAP-SP5 CAR ORF protein:

[0364] MTRLTVLALLAGLLASSRAHHHHHHGGGGSSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 26)

[0365] M(2339VHH) DNA sequence:

[0366] CAGCTGCAGCTGGGCGCCTCTGGCGGCGGCCTGGTCCAGCCTGGCGGCTCTCTGAGACTGAGCTGTGCCCTGTCTGGCTTCACACTGAGAGAGCTGGACGAGTTCGCCATCGGCTGGTTCAGGCAGGCCCCTGGCAAGGAGAGAGAGGGCGTGAGCTGTATCAGCGGCACAGGCGGCATCACACATTATGCTGACAGCGTGAAGGGCAGGTTCACAATCAGCAGAGACATCGCCAAGACAACCGTGTACCTGCAGATGAATAGCCTGAACAGCGAAGACACAGCCGTGTACTACTGTGCCGCCGACGAGAGATGTACAGACAGACTGATCAGACCTCCTACATATTGGGGACAAGGCACCCAGGTGACAGTCTCTTCT (SEQ ID NO: 27)

[0367] M(2339VHH) protein sequence: QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSS (SEQ ID NO: 28)

[0368] BCMA B029 (VHH) sequence in TCP001-C and MC001C:

[0369] QVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSS (SEQ ID NO: 29)

[0370] CD19 scFv sequence in TCP011-P (from CN201480027401.4):

[0371] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGG GGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 30)

[0372] EGFR E454 (VHH) sequence in TCP021-P:

[0373] QVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSS (SEQ ID NO: 31)

[0374] GFP scFv sequence in TCP001-N:

[0375] QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS (SEQ ID NO: 32)

[0376] Anti-TGFp scF from US7494651B2 (mAb 12.7):

[0377] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSEWMNWVRQAPGQGLEWMGQIFPALGSTNYNEMYEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGIGNYALDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDFYGNSFMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNIEDPLTFGGGTKVEIK (SEQ ID NO: 33)

[0378] PD-L1 BMK1 VHH in US20180327494 (Nivolumab): QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS (SEQ ID NO: 34)

[0379] 1444 (VHH) protein sequence:

[0380] QVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTGMYYCAASNKDYNDYDPDWGQGTQVTVSS (SEQ ID NO: 35)

[0381] B2121 scFv sequence in TCP001-P:

[0382] DIVLTQSPASLAMSLGERATISCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISRVQAEDAAIYSCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGESVKISCKASGYTFTDYSINWVKQAPGQGLKWMGWINTETREPAYAYDFRGRFVFSLDTSASTAYLQISSLKAEDTAVYFCALDYSYAMDYWGQGTLVTVSS (SEQ ID NO: 36)

[0383] TCP001-C:

[0384] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 37)

[0385] TCP001-P:

[0386] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAMSLGERATISCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISRVQAEDAAIYSCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGESVKISCKASGYTFTDYSINWVKQAPGQGLKWMGWINTETREPAYAYDFRGRFVFSLDTSASTAYLQISSLKAEDTAVYFCALDYSYAMDYWGQGTLVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 38)

[0387] TCP001-N:

[0388] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 39)

[0389] TCP011-P:

[0390] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 40)

[0391] TCP021-P:

[0392] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLNLSCAASGFDFSSVTMSWHRQSPGKERETVAVISNIGNRNVGSSVRGRFTISRDNKKQTVHLQMDNLKPEDTGIYRCKAWGLDLWGPGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 41)

[0393] TCP002-C:

[0394] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSAAAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSS (SEQ ID NO: 42)

[0395] TCP003-C:

[0396] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSAAAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 43)

[0397] TCP-MC:

[0398] QVQLVESGGGLVQPGGSLRLSCAASGSITSIYAIGWYRQAPGKLRELVAAITTSGNTFYRDSVKGRFTISRDNAKNTVSLQMNSLKSEDTAVYDCNGAPWGDHAPLVVSWDQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 44) TCP-MD:

[0399] QLQLGASGGGLVQPGGSLRLSCALSGFTLRELDEFAIGWFRQAPGKEREGVSCISGTGGITHYADSVKGRFTISRDIAKTTVYLQMNSLNSEDTAVYYCAADERCTDRLIRPPTYWGQGTQVTVSSGGGGSEQKLISEEDLGGGGSHHHHHH (SEQ ID NO: 45)

[0400] Having shown and described exemplary implementations of the subject matter contained herein, further changes and modifications can be made to the methods and systems described herein without departing from the scope of the claims. Additionally, certain steps in the methods and steps described above can not have to be performed in the precise order described. Rather, the steps can be performed in any order or simultaneously, depending on the implementation desired. Accordingly, the application is intended to embrace all such alterations, modifications, and variations that fall within the scope of this disclosure, including those that are already known or those that are developed later. Some such modifications of this application will be readily apparent to those skilled in the art, i.e., with respect to the exemplary embodiments discussed above, geometries, materials, dimensions, ratios, steps, etc. Accordingly, the application is not to be limited to the specific details shown and described above. The claims should therefore not be limited to the structures and operations described in the specification and drawings. DETAILED DESCRIPTION

[0402] Embodiment 1 : An immune cell comprising an expressed immune cell activating polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular marker domain, wherein the immune cell secretes one or more polypeptide effector molecules.

[0403] Embodiment 2: An immune cell comprising an expressed immune cell activating polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular chimeric polypeptide comprising a binding domain of a VHH antibody or single chain variable fragment and a marker domain, wherein the immune cell secretes one or more polypeptide effector molecules.

[0404] Embodiment 3: The immune cell of embodiment 1 or embodiment 2, wherein the marker domain comprises a polypeptide derived from a structural membrane protein or a fetoprotein.

[0405] Embodiment 4: The immune cell of any one of embodiments 1-3, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds one or more immune modulators.

[0406] Embodiment 5: The immune cell of embodiment 4, wherein the antibody is a VHH antibody.

[0407] Embodiment 6: The immune cell of embodiment 4, wherein the immune modulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFp, TGFpR, HVEM, or LIGHT.

[0408] Embodiment 7: The immune cell of any one of embodiments 1-6, wherein the marker domain specifically binds a bi-specific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds a cell surface receptor of a cell.

[0409] Embodiment 8: An immune cell comprising a nucleic acid vector, the nucleic acid vector comprising:

[0410] (a) a promoter region operable for transcription in the immune cell;

[0411] (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide, the polypeptide comprising a signal transduction domain, a transmembrane domain, and a marker domain; and

[0412] (c) a terminator region operable to terminate transcription of the immune cell.

[0413] Embodiment 9: The immune cell of embodiment 8, further comprising a second nucleic acid vector, the second nucleic acid vector comprising:

[0414] (a) a promoter region operable for transcription in the immune cell;

[0415] (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules;

[0416] (c) a terminator region operable to terminate transcription of the immune cell.

[0417] Embodiment 10: The immune cell as in embodiment 8, wherein the nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.

[0418] Embodiment 11: The immune cell as in any one of embodiments 8-10, wherein the immune cell activating polypeptide further comprises a binding domain of a VHH antibody or single chain variable fragment.

[0419] Embodiment 12: The immune cell as in any one of embodiments 8-11, wherein the immune cell activating polypeptide comprises a chimeric polypeptide comprising (i) a binding domain of a VHH antibody or single chain variable fragment and (ii) the marker domain.

[0420] Embodiment 13: The immune cell as in embodiment 12, wherein the chimeric polypeptide is branched.

[0421] Embodiment 14: The immune cell as in any one of embodiments 8-13, wherein the marker domain comprises a polypeptide derived from a vitellogenin.

[0422] Embodiment 15: The immune cell as in any one of embodiments 8-13, wherein the marker domain comprises a structural membrane protein.

[0423] Embodiment 16: The immune cell as in any one of embodiments 8-15, wherein the signal transduction domain comprises a costimulatory domain and a T cell receptor (TCR) signaling domain.

[0424] Embodiment 17: The immune cell as in embodiment 16, wherein the costimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L.

[0425] Embodiment 18: The immune cell as in embodiment 16 or embodiment 17, wherein the TCR signaling domain comprises CD3 zeta or CD3 epsilon.

[0426] Embodiment 19: The immune cell as in any one of embodiments 16-18, wherein the signal transduction domain comprises CD28 and CD3 zeta.

[0427] Embodiment 20: The immune cell as in any one of embodiments 8-19, wherein the transmembrane domain comprises a domain involved in immune costimulatory signaling.

[0428] Embodiment 21: The immune cell as in any one of embodiments 8-20, wherein the transmembrane domain comprises CD28.

[0429] Embodiment 22: The immune cell of embodiment 21, wherein the CD28 comprises an ITAM domain.

[0430] Embodiment 23: The immune cell of any one of embodiments 8-18 and 20-22, wherein the CD3s domain comprises the amino acids YMNM.

[0431] Embodiment 24: The immune cell of any one of embodiments 8-23, wherein the at least one nucleic acid vector further comprises a PiggyBac transposase.

[0432] Embodiment 25: The immune cell of any one of embodiments 8-23, wherein the at least one nucleic acid vector further comprises a transposon inverted terminal repeat sequence.

[0433] Embodiment 26: The immune cell of any one of embodiments 8-25, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds to one or more immunomodulators.

[0434] Embodiment 27: The immune cell of embodiment 26, wherein the antibody is a VHH antibody.

[0435] Embodiment 28: The immune cell of embodiment 26 or embodiment 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, TGFp, TGFpR, HVEM, or LIGHT.

[0436] Embodiment 28: The immune cell of any one of embodiments 8-25, wherein the polypeptide effector molecule comprises a cytokine.

[0437] Embodiment 30: The immune cell of embodiment 29, wherein the cytokine is TGF-p, VEGF, TNF-a, CCR5, CCR7, IL-2, IL-7, IL-15, or IL-17.

[0438] Embodiment 31: The immune cell of any one of embodiments 8-30, which is a T cell, a tumor infiltrating lymphocyte, a cytokine activated killer cell, a dendritic cell-cytokine activated killer cell, a gd-T cell, a natural killer T cell, or a natural killer cell.

[0439] Embodiment 32: An immune cell activator polypeptide, comprising:

[0440] (a) a marker domain;

[0441] (b) a transmembrane domain; and

[0442] (c) a signal transduction domain.

[0443] Embodiment 33: The immune cell activator polypeptide of embodiment 32, wherein the signal transduction domain comprises a costimulatory domain and a T cell receptor (TCR) signaling domain.

[0444] Embodiment 34: The immune cell activator polypeptide of embodiment 33, wherein the costimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L.

[0445] Embodiment 35: The immune cell activator polypeptide of embodiment 33, wherein the TCR signaling domain comprises CD3 zeta or CD3 epsilon.

[0446] Embodiment 36: The immune cell activator polypeptide of embodiment 33, wherein the signal transduction domain comprises CD28 linked at its C-terminus to the N-terminus of a CD3 epsilon signaling domain.

[0447] Embodiment 37: The immune cell activator polypeptide of embodiment 33, wherein the signal transduction domain comprises a costimulatory domain 4-1BB linked at its C-terminus to the N-terminus of a CD3 epsilon signaling domain.

[0448] Embodiment 38: The immune cell activator polypeptide of any one of embodiments 32-37, wherein the marker domain comprises a polypeptide derived from a vitellogenin.

[0449] Embodiment 39: The immune cell activator polypeptide of any one of embodiments 32-37, wherein the marker domain comprises a structural membrane protein.

[0450] Embodiment 40: The immune cell activator polypeptide of any one of embodiments 32-39, wherein the transmembrane domain comprises a domain involved in immune costimulatory signaling.

[0451] Embodiment 41: The immune cell activator polypeptide of any one of embodiments 32-40, wherein the transmembrane domain comprises CD28 or a structural membrane protein.

[0452] Embodiment 42: The immune cell activator polypeptide of any one of embodiments 32-41, wherein CD28 comprises an ITAM domain.

[0453] Embodiment 43: The immune cell activator polypeptide of any one of embodiments 32-42, wherein the CD3e domain comprises the amino acids YMNM.

[0454] Embodiment 44: A nucleic acid vector comprising

[0455] (a) a promoter region effective for transcription in an immune cell;

[0456] (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0457] (c) a terminator region effective to terminate transcription of the immune cell.

[0458] Embodiment 45: The nucleic acid vector of embodiment 44, further comprising a transposon inverted terminal repeat sequence.

[0459] Embodiment 46: A nucleic acid vector comprising:

[0460] (a) a promoter region effective for transcription in an immune cell;

[0461] (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.

[0462] (c) a terminator region effective to terminate transcription of the immune cell.

[0463] Embodiment 47: The nucleic acid vector of embodiment 46, further comprising a transposon inverted terminal repeat sequence.

[0464] Embodiment 48: The nucleic acid vector of embodiment 46 or embodiment 47, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds one or more immunomodulatory agents.

[0465] Embodiment 49: The nucleic acid vector of embodiment 48, wherein the antibody is a VHH antibody.

[0466] Embodiment 50: The nucleic acid vector of embodiment 46 or embodiment 47, wherein the polypeptide effector molecule comprises a cytokine.

[0467] Embodiment 51: A bispecific polypeptide comprising:

[0468] (a) a label-binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a label domain of the immune cell activator polypeptide of any one of embodiments 32-40; and

[0469] (b) a cell surface protein-binding domain (CSP-bd) comprising a single chain polypeptide domain that binds to a cell surface receptor of a cell.

[0470] Embodiment 52: The bispecific polypeptide of embodiment 51, wherein the marker binding domain comprises a VHH domain of a camelid IgG.

[0471] Embodiment 53: The bispecific polypeptide of embodiment 51 or embodiment 52, comprising a CDR3 domain of about 15-20 amino acids.

[0472] Embodiment 54: The bispecific polypeptide of any one of embodiments 51-53, wherein the cell is a lymphocyte.

[0473] Embodiment 55: The bispecific polypeptide of embodiment 54, wherein the lymphocyte is a B cell.

[0474] Embodiment 56: The bispecific polypeptide of any one of embodiments 51-53, wherein the cell is a tumor cell.

[0475] Embodiment 57: The bispecific polypeptide of embodiment 56, wherein the tumor is a lymphoma, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, or mesothelioma.

[0476] Embodiment 58: The bispecific polypeptide of embodiment 56 or 57, wherein the cell surface protein is EGFR.

[0477] Embodiment 59: The bispecific polypeptide of embodiment 56 or 57, wherein the cell surface protein is GPC3.

[0478] Embodiment 60: The bispecific polypeptide of any one of embodiments 51-57, wherein the cell surface protein binding domain specifically binds to an EGFR protein expressed on the surface of a tumor cell.

[0479] Embodiment 61: The bispecific polypeptide of any one of embodiments 51-57, wherein the cell surface protein binding domain specifically binds to CD19, CD20, or CD22 on the surface of a lymphoma cell.

[0480] Embodiment 62: The bispecific polypeptide of any one of embodiments 51-57, comprising a VHH antibody.

[0481] Embodiment 63: The bispecific polypeptide of any one of embodiments 51-62, further comprising one or more domains that provide an additional biochemical activity or biological function.

[0482] Embodiment 64: The bispecific polypeptide of embodiment 63, wherein the additional biochemical activity or biological function comprises: specific binding of a fluorophore, prolonging the in vivo half-life of the bispecific polypeptide, increasing the affinity of the bispecific polypeptide, and modulating an immune response mediated by the Fc domain.

[0483] Embodiment 65: The bispecific polypeptide of any one of embodiments 51-62, further comprising an additional cell surface protein binding domain comprising a single chain polypeptide domain that binds a different cell surface receptor of the same or a different cell.

[0484] Embodiment 66: A kit for producing one or more polypeptide effector molecules in situ at the location of a target cell, comprising:

[0485] (a) an immune cell of any one of embodiments 8-31; and

[0486] (b) a bispecific polypeptide of any one of embodiments 51-65.

[0487] Embodiment 67: The kit of embodiment 66, wherein the cell surface protein binding domain specifically binds CD19 on a B cell.

[0488] Embodiment 68: The kit of embodiment 66 or embodiment 67, wherein the cell surface protein binding domain specifically binds EGFR, mesothelin, BCMA, MUC1, or GPC3 on a tumor cell.

[0489] Embodiment 69: A method of modulating the immune system environment at the location of a tumor cell in a subject, comprising:

[0490] (a) administering to the subject an effective amount of an immune cell of any one of embodiments 9-31 and an effective amount of a first bispecific polypeptide of any one of embodiments 51-65 simultaneously or sequentially, wherein the bispecific polypeptide comprises a cell surface protein binding domain that specifically binds a cell surface protein of a lymphocyte; and

[0491] (b) administering to the subject an effective amount of a second bispecific polypeptide of any one of embodiments 51-65, wherein the bispecific polypeptide comprises a cell surface protein binding domain that specifically binds a cell surface protein of a tumor cell.

[0492] Embodiment 70: The method of embodiment 69, further comprising a step of measuring the amount of immune cells in the subject performed between steps a and b.

[0493] Embodiment 71 : The method of embodiment 70, wherein the amount of immune cells in the blood of the subject is measured.

[0494] Embodiment 72: The method of embodiment 70, wherein the number of immune cells infiltrating a tumor of the subject is measured.

[0495] Embodiment 73: The method of any one of embodiments 69-72, wherein the immune cells are T cells, tumor infiltrating lymphocytes, cytokine activated killer cells, dendritic cell-cytokine activated killer cells, gamma delta-T cells, natural killer T cells, or natural killer cells.

[0496] Embodiment 74: The method of any one of embodiments 69-73, wherein the cell surface protein of the lymphocyte is CD19 of a B cell.

[0497] Embodiment 75: The method of any one of embodiments 69-74, wherein the tumor cell is a lymphoma cell, mesothelial cell, non-small cell lung cancer cell, ovarian cell, hepatocarcinoma or breast cancer cell.

[0498] Embodiment 76: The method of embodiment 75, wherein the cell surface protein is EGFR, mesothelin, BCMA, MUC1 or GPC3.

[0499] Embodiment 77: A method of modulating an immune system environment at a location of a tumor cell in a subject, comprising:

[0500] (a) propagating a transformed immune cell of the subject in vitro to obtain a propagated T cell; and administering the propagated T cell to the subject; wherein the immune cell comprises a first nucleic acid vector, the first nucleic acid vector comprising a nucleic acid vector comprising:

[0501] (i) a promoter region operable for transcription in an immune cell;

[0502] (ii) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide; and

[0503] (iii) a terminator region operable to terminate transcription in an immune cell;

[0504] and the immune cell comprises a second nucleic acid vector, the second nucleic acid vector comprising:

[0505] (iv) a promoter region operable for transcription in an immune cell;

[0506] (v) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; and

[0507] (vi) a terminator region effective to terminate transcription in the immune cell;

[0508] and

[0509] (b) administering to the subject an effective amount of a bispecific polypeptide to activate proliferating immune cells to express an immunomodulatory polypeptide, the bispecific polypeptide comprising a cell surface protein binding domain and a marker binding domain having a defined amino acid sequence, the marker binding domain specifically binds to a marker domain expressed by the proliferating immune cells, the cell surface protein binding domain specifically binds to a cell surface receptor of a tumor cell.

[0510] Embodiment 78: The method of embodiment 77, wherein the tumor cell is a mesothelial cell that overexpresses mesothelin and PDL1, and the cell surface protein is mesothelin expressed on the surface of the mesothelial cell, and wherein the effector molecule comprises a VHH domain that specifically binds PD-1 or CD40.

[0511] Embodiment 79: The method of embodiment 77 or embodiment 78, wherein the tumor cell is a B cell, and the cell surface protein is CD19, CD20, or CD22 on the surface of the B cell.

[0512] Embodiment 80: The method of any one of embodiments 77-79, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte, a cytokine activated killer cell, a dendritic cell-cytokine activated killer cell, a gamma delta-T cell, a natural killer T cell, or a natural killer cell. SEQUENCE LISTING <110> Shanghai Cell Therapy Group Co., Ltd. Shanghai Cell Therapy Group Pharmaceutical Technology Co., Ltd. <120> Cells expressing immunomodulatory molecules and systems expressing immunomodulatory molecules <130> SHC001PCT <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Artificial <220> <223> Human signal peptide <400> 1 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser <210> 2 <211> 19 <212> PRT <213> Artificial <220> <223> Human signal peptide <400> 2 Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser 1 5 10 15 Ser Arg Ala <210> 3 <211> 123 <212> PRT <213> Vicugna pacos <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Ser Phe Ile Ser 20 25 30 Ala Ala Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Asn Thr Gly Ile Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Gly Ala Pro Pro Pro Gly Gly Leu Gly Tyr Asp Glu Ser Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 4 <212> PRT <213> Human <400> 4 Tyr Met Asn Met 1 <210> 5 <211> 60 <212> PRT <213> Human <400> 5 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly 1 5 10 15 Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro 20 25 30 Arg Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly 35 40 45 Pro Gly Val Gly Pro Gly Ser Glu Val Trp Gly Ile 50 55 60 <210> 6 <211> 53 <212> PRT <213> Human <400> 6 Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln 1 5 10 15 Thr Ser Gly Gly Gly Gly Gly Gly Asn Ser Thr Ala Ala Ala Ala Gly Gly 20 25 30 Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn Ala Phe 35 40 45 Met Val Trp Ser Arg 50 <210> 7 <211> 54 <212> PRT <213> Human <400> 7 Met Ala Leu Ser Glu Pro Ile Leu Pro Ser Phe Ser Thr Phe Ala Ser 1 5 10 15 Pro Cys Arg Glu Arg Gly Leu Gln Glu Arg Trp Pro Arg Ala Glu Pro 20 25 30 Glu Ser Gly Gly Thr Asp Asp Asp Leu Asn Ser Val Leu Asp Phe Ile 35 40 45 Leu Ser Met Gly Leu Asp 50 <210> 8 <211> 38 <212> PRT <213> Artificial <220> <223> Camelidae ICAP tag domain <400> 8 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly 1 5 10 15 Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro 20 25 30 Arg Thr Trp Leu Ser Phe 35 <210> 9 <211> 64 <212> PRT <213> Human <400> 9 Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys Ala Arg Glu Ile 1 5 10 15 Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val 20 25 30 Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro 35 40 45 Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu 50 55 60 <210> 10 <211> 96 <212> PRT <213> Human <400> 10 Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu 1 5 10 15 Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg 20 25 30 Gly Gin Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro 35 40 45 Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro 50 55 60 Ser Ser Ile Trp Ala Val Arg Pro Gin Asp Leu Asp Thr Cys Asp Pro 65 70 75 80 Arg Gin Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gin Asn 85 90 95 <210> 11 <211> 101 <212> PRT <213> Human <400> 11 Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser Ile 1 5 10 15 Trp Ala Val Arg Pro Gin Asp Leu Asp Thr Cys Asp Pro Arg Gin Leu 20 25 30 Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gin Asn Met Asn Gly 35 40 45 Ser Glu Tyr Phe Val Lys Ile Gin Ser Phe Leu Gly Gly Ala Pro Thr 50 55 60 Glu Asp Leu Lys Ala Leu Ser Gin Gin Asn Val Ser Met Asp Leu Ala 65 70 75 80 Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val Ala 85 90 95 Glu Val Gln Lys Leu 100 <210> 12 <211> 6 <212> PRT <213> Artificial <220> <223> Metalloprotease cleavable linker <400> 12 Arg Val Leu Ala Glu Ala 1 5 <210> 13 <211> 10 <212> PRT <213> Artificial <220> <223> Metalloprotease cleavable linker <400> 13 Glu Asp Val Val Cys Cys Ser Met Ser Tyr 1 5 10 <210> 14 <211> 8 <212> PRT <213> Artificial <220> <223> Metalloprotease cleavable linker <400> 14 Gly Gly Ile Glu Gly Arg Gly Ser 1 5 <210> 15 <211> 17 <212> PRT <213> Artificial <220> <223> Factor IXa / Factor VIIa cleavable linker <400> 15 Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 16 <211> 254 <212> PRT <213> Vicugna pacos <400> 16 Gln Leu Gin Leu Gly Ala Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gin Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 130 135 140 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Asn 145 150 155 160 Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Val Thr Met Ser 165 170 175 Trp His Arg Gln Ser Pro Gly Lys Glu Arg Glu Thr Val Ala Val Ile 180 185 190 Ser Asn Ile Gly Asn Arg Asn Val Gly Ser Ser Val Arg Gly Arg Phe 195 200 205 Thr Ile Ser Arg Asp Asn Lys Lys Gln Thr Val His Leu Gln Met Asp 210 215 220 Asn Leu Lys Pro Glu Asp Thr Gly Ile Tyr Arg Cys Lys Ala Trp Gly 225 230 235 240 Leu Asp Leu Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 245 250 <210> 17 <211> 54 <212> PRT <213> Human <400> 17 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala 50 <210> 18 <211> 23 <212> PRT <213> Human <400> 18 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro 20 <210> 19 <211> 624 <212> DNA <213> Human <400> 19 tccctggaga ccctgaaggc tttgcttgaa gtcaacaaag ggcacgaaat gagtcctcag 60 gtggccaccc tgatcgaccg ctttgtgaag ggaaggggcc agctagacaa agacacccta 120 gacaccctga ccgccttcta ccctgggtac ctgtgctccc tcagccccga ggagctgagc 180 tccgtgcccc ccagcagcat ctgggcggtc aggccccagg acctggacac gtgtgaccca 240 tccgtgcccc ccagcagcat ctgggcggtc aggccccagg acctggacac gtgtgaccca 240aggcagctgg acgtcctcta tcccaaggcc cgccttgctt tccagaacat gaacgggtcc 300 gaatacttcg tgaagatcca gtccttcctg ggtggggccc ccacggagga tttgaaggcg 360 ctcagtcagc agaatgtgag catggacttg gccacgttca tgaagctgcg gacggatgcg 420 gtgctgccgt tgactgtggc tgaggtgcag aaacttctgg gaccccacgt ggagggcctg 480 aaggcggagg agcggcaccg cccggtgcgg gactggatcc tacggcagcg gcaggacgac 540 ctggacacgc tggggctggg gctacagggc ggcatcccca acggctacct ggtcctagac 600 ctcagcatgc aagaggccct ctcg 624 <210> 20 <211> 208 <212> PRT <213> Human <400> 20 Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu 1 5 10 15 Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg 20 25 30 Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro 35 40 45 Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro 50 55 60 Ser Ser Ile Trp Ala Val Arg Pro Gin Asp Leu Asp Thr Cys Asp Pro 65 70 75 80 Arg Gin Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gin Asn 85 90 95 Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gin Ser Phe Leu Gly Gly 100 105 110 Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gin Gin Asn Val Ser Met 115 120 125 Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu 130 135 140 Thr Val Ala Glu Val Gin Lys Leu Leu Gly Pro His Val Glu Gly Leu 145 150 155 160 Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gin 165 170 175 Arg Gin Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gin Gly Gly Ile 180 185 190 Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gin Glu Ala Leu Ser 195 200 205 <210> 21 <211> 1293 <212> DNA <213> Artificial <220> <223> M-ICAP CAR ORF-encoding DNA <400> 21 atggccttgc caacggctcg acccctgttg gggtcctgtg ggacccccgc cctcggcagc 60 ctcctgttcc tgctcttcag cctcggatgg gtgcagcccc accaccacca tcaccacgga 120 ggaggcggat cttccctgga gaccctgaag gctttgcttg aagtcaacaa agggcacgaa 180 atgagtcctc aggtggccac cctgatcgac cgctttgtga agggaagggg ccagctagac 240 aaagacaccc tagacaccct gaccgccttc taccctgggt acctgtgctc cctcagcccc 300 gaggagctga gctccgtgcc ccccagcagc atctgggcgg tcaggcccca ggacctggac 360 acgtgtgacc caaggcagct ggacgtcctc tatcccaagg cccgccttgc tttccagaac 420 atgaacgggt ccgaatactt cgtgaagatc cagtccttcc tgggtggggc ccccacggag 480 gatttgaagg cgctcagtca gcagaatgtg agcatggact tggccacgtt catgaagctg 540 cggacggatg cggtgctgcc gttgactgtg gctgaggtgc agaaacttct gggaccccac 600 gtggagggcc tgaaggcgga ggagcggcac cgcccggtgc gggactggat cctacggcag 660 cggcaggacg acctggacac gctggggctg gggctacagg gcggcatccc caacggctac 720 ctggtcctag acctcagcat gcaagaggcc ctctcgatct acatctgggc gcccctggcc 780 gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 840 aagaagctcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 900 gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 960 aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1020 gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1080 cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1140 cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1200 ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1260 gcccttcaca tgcaggccct gccccctcgc tga 1293 <210> 22 <211> 430 <212> PRT <213> Artificial <220>; <223> M-ICAP CAR ORF Proteins <400> 22 Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro His His His His His His Gly Gly Gly Gly Ser Ser Leu Glu Thr 35 40 45 Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met Ser Pro Gln 50 55 60 Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln Leu Asp 65 70 75 80 Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys 85 90 95 Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser Ile Trp 100 105 110 Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp 115 120 125 Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn Gly Ser 130 135 140 Glu Tyr Phe Val Lys lie Gin Ser Phe Leu Gly Gly Ala Pro Thr Glu 145 150 155 160 Asp Leu Lys Ala Leu Ser Gin Gin Asn Val Ser Met Asp Leu Ala Thr 165 170 175 Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu 180 185 190 Val Gin Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu 195 200 205 Arg His Arg Pro Val Arg Asp Trp lie Leu Arg Gin Arg Gin Asp Asp 210 215 220 Leu Asp Thr Leu Gly Leu Gly Leu Gin Gly Gly lie Pro Asn Gly Tyr 225 230 235 240 Leu Val Leu Asp Leu Ser Met Gin Glu Ala Leu Ser lie Tyr lie Trp 245 250 255 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val lie 260 265 270 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr lie Phe Lys 275 280 285 Gln Pro Phe Met Arg Pro Val Gin Thr Thr Gin Glu Glu Asp Gly Cys 290 295 300 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 305 310 315 320 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 325 330 335 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 340 345 350 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 355 360 365 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 370 375 380 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 385 390 395 400 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 405 410 415 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 420 425 430 <210> 23 <211> 1251 <212> DNA <213> Artificial <220> <223> M-ICAP-SP3 CAR ORF-encoding DNA <400> 23 atgaagcacc tctggttctt cctcctgctg gtggcagctc ctagatgggt gctgtctcac 60 caccaccatc accacggagg aggcggatct tccctggaga ccctgaaggc tttgcttgaa 120 gtcaacaaag ggcacgaaat gagtcctcag gtggccaccc tgatcgaccg ctttgtgaag 180 ggaaggggcc agctagacaa agacacccta gacaccctga ccgccttcta ccctgggtac 240 ctgtgctccc tcagccccga ggagctgagc tccgtgcccc ccagcagcat ctgggcggtc 300 aggccccagg acctggacac gtgtgaccca aggcagctgg acgtcctcta tcccaaggcc 360 cgccttgctt tccagaacat gaacgggtcc gaatacttcg tgaagatcca gtccttcctg 420 ggtggggccc ccacggagga tttgaaggcg ctcagtcagc agaatgtgag catggacttg 480 gccacgttca tgaagctgcg gacggatgcg gtgctgccgt tgactgtggc tgaggtgcag 540 aaacttctgg gaccccacgt ggagggcctg aaggcggagg agcggcaccg cccggtgcgg 600 gactggatcc tacggcagcg gcaggacgac ctggacacgc tggggctggg gctacagggc 660 ggcatcccca acggctacct ggtcctagac ctcagcatgc aagaggccct ctcgatctac 720 atctgggcgc ccctggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 780 tactgcaaac ggggcagaaa gaagctcctg tatatattca aacaaccatt tatgagacca 840 gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 900 ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 960 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1020 aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1080 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1140 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1200 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgctg a 1251 <210> 24 <211> 416 <212> PRT <213> Artificial <220> <223> M-ICAP-SP3 CAR protein <400> 24 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser His His His His His His His Gly Gly Gly Gly Ser Ser Leu 20 25 30 Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met Ser 35 40 45 Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln 50 55 60 Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr 65 70 75 80 Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser 85 90 95 Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln 100 105 110 Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn 115 120 125 Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro 130 135 140 Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu 145 150 155 160 Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val 165 170 175 Ala Glu Val Gin Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala 180 185 190 Glu Glu Arg His Arg Pro Val Arg Asp Trp He Leu Arg Gin Arg Gin 195 200 205 Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gin Gly Gly He Pro Asn 210 215 220 Gly Tyr Leu Val Leu Asp Leu Ser Met Gin Glu Ala Leu Ser He Tyr 225 230 235 240 He Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 245 250 255 Val He Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr He 260 265 270 Phe Lys Gin Pro Phe Met Arg Pro Val Gin Thr Thr Gin Glu Glu Asp 275 280 285 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 290 295 300 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gin Gin Gly 305 310 315 320 Gln Asn Gin Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 325 330 335 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 340 345 350 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 355 360 365 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 370 375 380 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 385 390 395 400 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 405 410 415 <210> 25 <211> 1251 <212> DNA <213> Artificial <220> <223>

[00245] M-ICAP-SP5 CAR ORF - encoding DNA <400> 25 atgaccaggc tgacagtgct ggctctgctg gccggactgc tggcttcttc tagagctcac 60 caccaccatc accacggagg aggcggatct tccctggaga ccctgaaggc tttgcttgaa 120 gtcaacaaag ggcacgaaat gagtcctcag gtggccaccc tgatcgaccg ctttgtgaag 180 ggaaggggcc agctagacaa agacacccta gacaccctga ccgccttcta ccctgggtac 240 ctgtgctccc tcagccccga ggagctgagc tccgtgcccc ccagcagcat ctgggcggtc 300 aggccccagg acctggacac gtgtgaccca aggcagctgg acgtcctcta tcccaaggcc 360 cgccttgctt tccagaacat gaacgggtcc gaatacttcg tgaagatcca gtccttcctg 420 ggtggggccc ccacggagga tttgaaggcg ctcagtcagc agaatgtgag catggacttg 480 gccacgttca tgaagctgcg gacggatgcg gtgctgccgt tgactgtggc tgaggtgcag 540 aaacttctgg gaccccacgt ggagggcctg aaggcggagg agcggcaccg cccggtgcgg 600 gactggatcc tacggcagcg gcaggacgac ctggacacgc tggggctggg gctacagggc 660 ggcatcccca acggctacct ggtcctagac ctcagcatgc aagaggccct ctcgatctac 720 atctgggcgc ccctggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 780 tactgcaaac ggggcagaaa gaagctcctg tatatattca aacaaccatt tatgagacca 840 gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 900 ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 960 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1020 aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1080 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1140 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1200 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgctg a 1251 <210> 26 <211> 416 <212> PRT <213> Artificial <220> <223> M-ICAP-SP5 CAR ORF Protein <400> 26 Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser 1 5 10 15 Ser Arg Ala His His His His His His Gly Gly Gly Gly Ser Ser Leu 20 25 30 Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met Ser 35 40 45 Pro Gin Val Ala Thr Leu lie Asp Arg Phe Val Lys Gly Arg Gly Gin 50 55 60 Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr 65 70 75 80 Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser 85 90 95 Ile Trp Ala Val Arg Pro Gin Asp Leu Asp Thr Cys Asp Pro Arg Gin 100 105 110 Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gin Asn Met Asn 115 120 125 Gly Ser Glu Tyr Phe Val Lys lie Gin Ser Phe Leu Gly Gly Ala Pro 130 135 140 Thr Gin Asp Leu Lys Ala Leu Ser Gin Gin Asn Val Ser Met Asp Leu 145 150 155 160 Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val 165 170 175 Ala Gin Val Gin Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala 180 185 190 Glu Glu Arg His Arg Pro Val Arg Asp Trp lie Leu Arg Gin Arg Gin 195 200 205 Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn 210 215 220 Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Ile Tyr 225 230 235 240 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 245 250 255 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 260 265 270 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 275 280 285 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 290 295 300 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 305 310 315 320 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 325 330 335 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 340 345 350 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 355 360 365 Asp Lys Met Ala Glu Ala Tyr Ser Glu lie Gly Met Lys Gly Glu Arg 370 375 380 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gin Gly Leu Ser Thr Ala 385 390 395 400 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gin Ala Leu Pro Pro Arg 405 410 415 <210> 27 <211> 378 <212> DNA <213> Artificial <220> <223> M(2339VHH) DNA Sequence <400> 27 cagctgcagc tgggcgcctc tggcggcggc ctggtccagc ctggcggctc tctgagactg 60 agctgtgccc tgtctggctt cacactgaga gagctggacg agttcgccat cggctggttc 120 aggcaggccc ctggcaagga gagagagggc gtgagctgta tcagcggcac aggcggcatc 180 acacattatg ctgacagcgt gaagggcagg ttcacaatca gcagagacat cgccaagaca 240 accgtgtacc tgcagatgaa tagcctgaac agcgaagaca cagccgtgta ctactgtgcc 300 gccgacgaga gatgtacaga cagactgatc agacctccta catattgggg acaaggcacc 360 caggtgacag tctcttct 378 <210> 28 <211> 126 <212> PRT <213> Artificial <220> <223> M(2339VHH) protein sequence <400> 28 Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 29 <211> 120 <212> PRT <213> Artificial <220> <223>

[00249] TCP001-C MC001C BCMA B029(VH) sequence <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Thr Ser Ile Tyr 20 25 30 Ala Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Thr Ser Gly Asn Thr Phe Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Asp Cys Asn 85 90 95 Gly Ala Pro Trp Gly Asp His Ala Pro Leu Val Val Ser Trp Asp Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 30 <211> 244 <212> PRT <213> Artificial <220> <223> TCP011-P CD19 scFv sequence <400> 30 Asp Ile Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gin Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gin 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Lys Ala Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu 115 120 125 Gln Gin Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser Val 130 135 140 Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp 145 150 155 160 Ile Arg Gin Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 165 170 175 Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr 180 185 190 Ile Ile Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser 195 200 205 Leu Gin Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr 210 215 220 Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser Val 225 230 235 240 Thr Val Ser Ser <210> 31 <211> 113 <212> PRT <213> Artificial <220> <223> TCP021-PEGFR E454 (VHH) <400> 31 Gln Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Val 20 25 30 Thr Met Ser Trp His Arg Gin Ser Pro Gly Lys Glu Arg Glu Thr Val 35 40 45 Ala Val Ile Ser Asn Ile Gly Asn Arg Asn Val Gly Ser Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Lys Lys Gln Thr Val His Leu 65 70 75 80 Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Gly Ile Tyr Arg Cys Lys 85 90 95 Ala Trp Gly Leu Asp Leu Trp Gly Pro Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 32 <211> 115 <212> PRT <213> Artificial <220> <223> TCP001-N GFP scFv sequence <400> 32 Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr 20 25 30 Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 33 <211> 247 <212> PRT <213> Artificial <220> <223> Anti-TGFB scF <400> 33 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Glu 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr 50 55 60 Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly lie Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Arg 145 150 155 160 Ala Ser Glu Ser Val Asp Phe Tyr Gly Asn Ser Phe Met His Trp Tyr 165 170 175 Gln Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Leu Ala Ser 180 185 190 Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gin Gin Asn lie Glu Asp Pro Leu Thr Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Glu lie Lys 245 <210> 34 <211> 128 <212> PRT <213> Vicugna pacos <400> 34 Gln Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg 20 25 30 Cys Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Arg Val 35 40 45 Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser 100 105 110 Gly Ala Phe Gin Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 35 <211> 119 <212> PRT <213> Vicuna (vicugna pacos) <400> 35 Gln Val Gln Val Val Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Pro Ile Ile Ser Ile Ala 20 25 30 Tyr Met Gly Trp Tyr Arg Gln Ile Ser Glu Lys Glu Arg Gln Leu Val 35 40 45 Ala Thr Ile Asn Ser Gly Gly Lys Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Met Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala 85 90 95 Ala Ser Asn Lys Asp Tyr Asn Asp Tyr Asp Pro Asp Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 36 <211> 246 <212> PRT <213> Vicuna (vicugna pacos) <400> 36 Asp Ile Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Met Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile 20 25 30 Gly Ala His Leu Ile His Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Val Gin Ala Glu Asp Ala Ala Ile Tyr Ser Cys Leu Gin Ser Arg 85 90 95 Ile Phe Pro Arg Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 115 120 125 Gly Gin Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly 130 135 140 Glu Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 145 150 155 160 Tyr Ser lie Asn Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Lys Trp 165 170 175 Met Gly Trp lie Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp 180 185 190 Phe Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala 195 200 205 Tyr Leu Gin lie Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe 210 215 220 Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser 245 <210> 37 <211> 287 <212> PRT <213> Vicugna pacos <400> 37 Gln Leu Gin Leu Gly Ala Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala lie Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys lie Ser Gly Thr Gly Gly lie Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp lie Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gin Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu lie Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin 130 135 140 Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg 145 150 155 160 Leu Ser Cys Ala Ala Ser Gly Ser lie Thr Ser lie Tyr Ala lie Gly 165 170 175 Trp Tyr Arg Gin Ala Pro Gly Lys Leu Arg Glu Leu Val Ala Ala lie 180 185 190 Thr Thr Ser Gly Asn Thr Phe Tyr Arg Asp Ser Val Lys Gly Arg Phe 195 200 205 Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu Gin Met Asn 210 215 220 Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Asp Cys Asn Gly Ala Pro 225 230 235 240 Trp Gly Asp His Ala Pro Leu Val Val Ser Trp Asp Gin Gly Thr Gin 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Gin Lys Leu lie Ser 260 265 270 Glu Glu Asp Leu Gly Gly Gly Gly Ser His His His His His His His 275 280 285 <210> 38 <211> 413 <212> PRT <213> Vicugna pacos <400> 38 Gln Leu Gin Leu Gly Ala Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala lie Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys lie Ser Gly Thr Gly Gly lie Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 130 135 140 Leu Thr Gln Ser Pro Ala Ser Leu Ala Met Ser Leu Gly Glu Arg Ala 145 150 155 160 Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile Gly Ala His 165 170 175 Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 180 185 190 Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala Arg Phe Ser 195 200 205 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Gln 210 215 220 Ala Glu Asp Ala Ala Ile Tyr Ser Cys Leu Gin Ser Arg Ile Phe Pro 225 230 235 240 Arg Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser 245 250 255 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gin Val 260 265 270 Gln Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Glu Ser Val 275 280 285 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Ser Ile 290 295 300 Asn Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Lys Trp Met Gly Trp 305 310 315 320 Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe Arg Gly 325 330 335 Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr Leu Gin 340 345 350 Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Leu 355 360 365 Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr 370 375 380 Val Ser Ser Gly Gly Gly Gly Ser Glu Gin Lys Leu lie Ser Glu Glu 385 390 395 400 Asp Leu Gly Gly Gly Gly Ser His His His His His His 405 410 <210> 39 <211> 282 <212> PRT <213> Vicugna pacos <400> 39 Gln Leu Gin Leu Gly Ala Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala lie Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys lie Ser Gly Thr Gly Gly lie Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp lie Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gin Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu lie Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin 130 135 140 Leu Val Glu Ser Gly Gly Ala Leu Val Gin Pro Gly Gly Ser Leu Arg 145 150 155 160 Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr Ser Met Arg 165 170 175 Trp Tyr Arg Gin Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly Met 180 185 190 Ser Ser Ala Gly Asp Arg Ser Ser Tyr Gin Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr He Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gin Met 210 215 220 Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn 225 230 235 240 Val Gly Phe Glu Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 245 250 255 Gly Gly Gly Gly Ser Gin Val Gin Gin Val Ser Val Ser Ser 260 265 270 Gly Gly Gly Ser His His His His His His 275 280 <210> 40 <211> 411 <212> PRT <213> Vicugna pacos <400> 40 Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin 130 135 140 Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val 145 150 155 160 Thr lie Ser Cys Arg Ala Ser Gin Asp lie Ser Lys Tyr Leu Asn Trp 165 170 175 Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu lie Tyr His Thr 180 185 190 Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Tyr Ser Leu Thr lie Ser Asn Leu Glu Gin Glu Asp lie 210 215 220 Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Tyr Thr Phe Gly 225 230 235 240 Gly Gly Thr Lys Leu Glu lie Thr Lys Ala Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gin Glu Ser 260 265 270 Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser Val Thr Cys Thr 275 280 285 Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 290 295 300 Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 305 310 315 320 Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 325 330 335 Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr 340 345 350 Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly ​​​​​​​​​​​​​​​​​​​​​​​​Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 130 135 140 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Asn 145 150 155 160 Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Val Thr Met Ser 165 170 175 Trp His Arg Gln Ser Pro Gly Lys Glu Arg Glu Thr Val Ala Val Ile 180 185 190 Ser Asn Ile Gly Asn Arg Asn Val Gly Ser Ser Val Arg Gly Arg Phe 195 200 205 Thr Ile Ser Arg Asp Asn Lys Lys Gln Thr Val His Leu Gln Met Asp 210 215 220 Asn Leu Lys Pro Glu Asp Thr Gly Ile Tyr Arg Cys Lys Ala Trp Gly 225 230 235 240 Leu Asp Leu Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser Gly Gly 245 250 255 Gly Gly Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly 260 265 270 Gly Ser His His His His His His 275 280 <210> 42 <211> 498 <212> PRT <213> Alpaca (vicugna pacos) <400> 42 Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 It should be noted that there is a possible error in the "羊驼(vicugna pacos)" in the original text. It might be better to use the more common term "alpaca" directly. The above translation is adjusted accordingly. Also, there is a potential error in the "0002342" in the original text which is likely a miswriting of "0002342", and the translation is based on the corrected form.Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gin Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Ala Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 ​​​​​​​​​​​​​​​​​​​Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu 260 265 270 Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser 355 360 365 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu 370 375 380 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 385 390 395 400 Ala Ala Ser Gly Ser Ile Thr Ser Ile Tyr Ala Ile Gly Trp Tyr Arg 405 410 415 Gln Ala Pro Gly Lys Leu Arg Glu Leu Val Ala Ala Ile Thr Thr Ser 420 425 430 Gly Asn Thr Phe Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 435 440 445 Arg Asp Asn Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys 450 455 460 Ser Glu Asp Thr Ala Val Tyr Asp Cys Asn Gly Ala Pro Trp Gly Asp 465 470 475 480 His Ala Pro Leu Val Val Ser Trp Asp Gln Gly Thr Gln Val Thr Val 485 490 495 Ser Ser <210> 43 <211> 402 <212> PRT <213> Vicugna pacos <400> 43 Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Ala Ala Ala Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 130 135 140 Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr 145 150 155 160 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 165 170 175 Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 180 185 190 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 195 200 205 Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 210 215 220 Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 260 265 270 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser He Thr Ser He Tyr 275 280 285 Ala Ile Gly Trp Tyr Arg Gin Ala Pro Gly Lys Leu Arg Glu Leu Val 290 295 300 Ala Ala Ile Thr Thr Ser Gly Asn Thr Phe Tyr Arg Asp Ser Val Lys 305 310 315 320 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu 325 330 335 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Asp Cys Asn 340 345 350 Gly Ala Pro Trp Gly Asp His Ala Pro Leu Val Val Ser Trp Asp Gin 355 360 365 Gly Thr Gin Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Gin Lys 370 375 380 Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly Gly Ser His His His His 385 390 395 400 His His <210> 44 <211> 146 <212> PRT <213> Vicugna pacos <400> 44 Gln Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser lie Thr Ser lie Tyr 20 25 30 Ala lie Gly Trp Tyr Arg Gin Ala Pro Gly Lys Leu Arg Glu Leu Val 35 40 45 Ala Ala lie Thr Thr Ser Gly Asn Thr Phe Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Asp Cys Asn 85 90 95 Gly Ala Pro Trp Gly Asp His Ala Pro Leu Val Val Ser Trp Asp Gin 100 105 110 Gly Thr Gin Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Gin Lys 115 120 125 Leu lie Ser Glu Glu Asp Leu Gly Gly Gly Gly Ser His His His His 130 135 140 His His 145 <210> 45 <211> 152 <212> PRT <213> Vicugna pacos <400> 45 Gln Leu Gln Leu Gly Ala Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Phe Thr Leu Arg Glu Leu 20 25 30 Asp Glu Phe Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Ser Gly Thr Gly Gly Ile Thr His Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Thr 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala Asp Glu Arg Cys Thr Asp Arg Leu Ile Arg Pro 100 105 110 Pro Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly 130 135 140 Gly Ser His His His His His His 145 150

Claims

1. An immune cell comprising an immune cell activator polypeptide expressed by the immune cell, the polypeptide comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular marker domain, and wherein the immune cell secretes one or more polypeptide effector molecules, wherein the marker domain specifically binds to a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds to a cell surface receptor of a cell, wherein the marker domain comprises a structural inert domain from a human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19, and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO:

28.

2. An immune cell comprising an immune cell activator polypeptide expressed by the immune cell, the polypeptide comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular chimeric polypeptide comprising a binding domain of a VHH antibody or a single chain variable fragment and a marker domain, and wherein the immune cell secretes one or more polypeptide effector molecules, wherein the marker domain specifically binds to a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds to a cell surface receptor of a cell, wherein the marker domain comprises a structural inert domain from a human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19, and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO:

28.

3. The immune cell of claim 1 or 2, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds to one or more immune modulators.

4. The immune cell of claim 3, wherein the antibody is a VHH antibody.

5. The immune cell of claim 3, wherein the immune modulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFp, TGFpR, HVEM, or LIGHT.

6. An immune cell comprising a nucleic acid vector, the nucleic acid vector comprising (a) a promoter region operable for transcription in the immune cell; ​ (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide comprising a signal transduction domain, a transmembrane domain, and a marker domain, wherein the marker domain specifically binds to a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds to a cell surface receptor of a cell, wherein the marker domain comprises a structural inert domain from human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19, and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO: 28; and (c) a terminator region effective to terminate transcription in the immune cell.

7. The immune cell of claim 6, further comprising a second nucleic acid vector comprising (a) a promoter region effective for transcription in the immune cell; (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; (c) a terminator region effective to terminate transcription in the immune cell.

8. The immune cell of claim 6, wherein the nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.

9. The immune cell of claim 6, wherein the immune cell activator polypeptide further comprises a binding domain of a VHH antibody or single chain variable fragment.

10. The immune cell of claim 6, wherein the immune cell activator polypeptide comprises a chimeric polypeptide comprising (i) a binding domain of a VHH antibody or single chain variable fragment and (ii) the marker domain.

11. The immune cell of claim 10, wherein the chimeric polypeptide is branched.

12. The immune cell of claim 6, wherein, The signal transduction domain comprises a costimulatory domain and a T cell receptor (TCR) signaling domain.

13. The immune cell of claim 12, wherein the costimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L.

14. The immune cell of claim 12, wherein the TCR signaling domain comprises CD3 zeta or CD3 epsilon.

15. The immune cell of claim 12, wherein the signal transduction domain comprises CD28 and CD3 zeta.

16. The immune cell of any one of claims 6-15, wherein the transmembrane domain comprises a domain involved in immune costimulatory signaling.

17. The immune cell of any one of claims 6-15, wherein the transmembrane domain comprises CD28.

18. The immune cell of claim 17, wherein the CD28 comprises an ITAM domain.

19. The immune cell of claim 14, wherein the CD3 epsilon domain comprises the amino acids YMNM.

20. The immune cell of any one of claims 6-15, wherein the at least one nucleic acid vector further comprises a PiggyBac transposase.

21. The immune cell of any one of claims 6-15, wherein the at least one nucleic acid vector additionally comprises a transposon inverted terminal repeat sequence.

22. The immune cell of any one of claims 6-15, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds to one or more immunomodulators.

23. The immune cell of claim 22, wherein the antibody is a VHH antibody.

24. The immune cell of claim 22, 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, TGFp, TGFpR, HVEM, or LIGHT.

25. The immune cell of any one of claims 6-15, wherein the polypeptide effector molecule comprises a cytokine.

26. The immune cell of claim 25, wherein the cytokine is TGF-b, VEGF, TNF-a, CCR5, CCR7, IL-2, IL-7, IL-15, or IL17.

27. The immune cell of any one of claims 6-15, which is a T cell, a TIL cell, a cytokine-activated killer cell, a dendritic cell-cytokine-activated killer cell, a gd-T cell, an NKT cell, or a natural killer cell.

28. An immune cell activator polypeptide comprising: (a) a marker domain, wherein the marker domain specifically binds to a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds to a cell surface receptor of a cell, wherein the marker domain comprises a structurally inert domain from human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19, and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO: 28; (b) a transmembrane domain; and (c) a signaling domain.

29. The immune cell activator polypeptide of claim 28, wherein, The signaling domain comprises a costimulatory domain and a T cell receptor (TCR) signaling domain.

30. The immune cell activator polypeptide of claim 29, wherein the costimulatory domain comprises CD28, ICOS, CD27, 4-1BB, OX40, or CD40L.

31. The immune cell activator polypeptide of claim 29, wherein, The TCR signaling domain comprises CD3zeta or CD3epsilon.

32. The immune cell activator polypeptide of claim 31, wherein, The signaling domain comprises CD28 linked at the C-terminus to the N-terminus of a CD3epsilon signaling domain.

33. The immune cell activator polypeptide of claim 31, wherein, The signaling domain comprises the costimulatory domain 4-1BB linked at the C-terminus to the N-terminus of a CD3epsilon signaling domain.

34. The immune cell activator polypeptide of any one of claims 28-33, wherein the transmembrane domain comprises a domain involved in immune co-stimulatory signaling.

35. The immune cell activator polypeptide of any one of claims 28-33, wherein the transmembrane domain comprises CD28 or a structurally membrane protein.

36. The immune cell activator polypeptide of claim 35, wherein, the CD28 comprises an ITAM domain.

37. The immune cell activator polypeptide of any one of claims 31-33, wherein, the CD3 epsilon domain comprises the amino acids YMNM.

38. A nucleic acid vector comprising (a) a promoter region effective for transcription in an immune cell; (b) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide, wherein the immune cell activator polypeptide comprises an intracellular signal transduction domain, a transmembrane domain, and an extracellular marker domain, wherein the marker domain specifically binds a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds a cell surface receptor of a cell, wherein the marker domain comprises a structurally inert domain from human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19, and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO: 28; and (c) a terminator region effective to terminate transcription in an immune cell.

39. The nucleic acid vector of claim 38, further comprising a transposon inverted terminal repeat sequence.

40. A nucleic acid vector comprising (a) a promoter region effective for transcription in an immune cell, wherein the immune cell is the immune cell of any one of claims 6-27; (b) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; (c) a terminator region effective to terminate transcription in an immune cell.

41. The nucleic acid vector of claim 40, further comprising a transposon inverted terminal repeat sequence.

42. The nucleic acid vector of claim 40 or 41, wherein the polypeptide effector molecule comprises an antibody or binding fragment thereof that specifically binds to one or more immunomodulatory agents.

43. The nucleic acid vector of claim 42, wherein the antibody is a VHH antibody.

44. The nucleic acid vector of claim 40 or 41, wherein the polypeptide effector molecule comprises a cytokine.

45. A bispecific polypeptide comprising: (a) a marker binding domain (L-bd) comprising a single chain polypeptide domain that specifically binds to a marker domain of the immune cell activator polypeptide of any one of claims 28-37; and (b) a cell surface protein binding domain (CSP-bd) comprising a single chain polypeptide domain that binds to a cell surface receptor of a cell.

46. The bispecific polypeptide of claim 45, wherein the marker binding domain comprises a VHH domain of a camelid IgG.

47. The bispecific polypeptide of claim 45, comprising about 15-20 amino acids of a CDR3 domain.

48. The bispecific polypeptide of any one of claims 45-47, wherein the cell is a lymphocyte.

49. The bispecific polypeptide of claim 48, wherein the lymphocyte is a B cell.

50. The bispecific polypeptide of any one of claims 45-47, wherein the cell is a tumor cell.

51. The bispecific polypeptide of claim 50, wherein the tumor is a lymphoma, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, or mesothelioma.

52. The bispecific polypeptide of claim 50, wherein the cell surface protein is EGFR.

53. The bispecific polypeptide of any one of claims 45-47, wherein the cell surface protein binding domain specifically binds to an EGFR protein expressed on the surface of a tumor cell.

54. The bispecific polypeptide of any one of claims 45-47, wherein the cell surface protein binding domain specifically binds to CD19 on the surface of a lymphoma cell.

55. The bispecific polypeptide of any one of claims 45-47, comprising a VHH antibody.

56. The bispecific polypeptide of any one of claims 45-47, further comprising one or more domains that provide an additional biochemical activity or biological function.

57. The bispecific polypeptide of claim 56, wherein the additional biochemical activity or biological function comprises: specific binding of a fluorophore, prolonging the half-life of the bispecific polypeptide in vivo, increasing the affinity of the bispecific polypeptide, and modulating an immune response mediated by the Fc domain.

58. The bispecific polypeptide of any one of claims 45-47, further comprising an additional cell surface protein binding domain comprising a single chain polypeptide domain that can bind to a different cell surface receptor of the same or a different cell.

59. A kit for producing one or more polypeptide effector molecules in situ at the proximity of a target cell, comprising (a) the immune cell of any one of claims 6-27; and (b) the bispecific polypeptide of any one of claims 45-58.

60. The kit of claim 59, wherein the cell surface protein binding domain specifically binds to CD19 on a B cell.

61. The kit of claim 59 or 60, wherein the cell surface protein binding domain specifically binds to EGFR or BCMA on a tumor cell.

62. Use of a substance in the manufacture of a medicament for modulating the immune system environment at the location of a tumor cell in a subject, the substance being: (a) an effective amount of the immune cell of any one of claims 6-27 and an effective amount of a first bispecific polypeptide administered simultaneously or sequentially to the subject, wherein the first bispecific polypeptide is as claimed in any one of claims 45-58 and comprises a cell surface protein binding domain that specifically binds to a cell surface protein of a lymphocyte; and (b) an effective amount of a second bispecific polypeptide administered to the subject, wherein the second bispecific polypeptide is as claimed in any one of claims 45-58 and comprises a cell surface protein binding domain that specifically binds to a cell surface protein of a tumor cell.

63. The use of claim 62, wherein the compound is of formula (I): ###00017### (I) or a pharmaceutically acceptable salt thereof. The method further comprises a step of measuring the amount of immune cells in the subject performed between steps a and b.

64. The use of claim 63, wherein the compound is of formula (Ia) ###0010### (Ia). The step is measuring the amount of immune cells in the blood of the subject.

65. The use of claim 63, wherein the compound is ###0010### The step is measuring the amount of tumor infiltrating immune cells of the subject.

66. The use of any one of claims 62-65, wherein the antibody is administered at a dose of about 300 mg. Wherein the immune cells are T cells, TIL cells, cytokine activated killer cells, dendritic cell-cytokine activated killer cells, gd-T cells, NKT cells or natural killer cells.

67. Use according to any one of claims 62 to 65, wherein the compound is of formula (I) ###00017### (I) or a pharmaceutically acceptable salt thereof. Wherein the cell surface protein of the lymphocyte is CD19 of a B cell.

68. The use of any one of claims 62-65, wherein the antibody is administered at a dose of about 10 mg / kg. Wherein the tumor cells are lymphoma cells, mesothelioma cells, non-small cell lung cancer cells, ovarian cancer cells, liver cancer cells or breast cancer cells.

69. The use of claim 68, wherein the compound is of formula (Ia) ###0010### (Ia). Wherein the cell surface protein is EGFR or BCMA.

70. Use of a substance in the manufacture of a medicament for modulating the immune system environment at the location of tumor cells in a subject, the substance being: (a) a proliferated T cell obtained from proliferating transformed immune cells of the subject in vitro; wherein the immune cells comprise a first nucleic acid vector, the first nucleic acid vector comprising a nucleic acid vector comprising: (i) a promoter region operable for transcription in an immune cell; (ii) a polynucleotide encoding an amino acid sequence of an immune cell activator polypeptide, wherein the immune cell activator polypeptide comprises an intracellular signal transduction domain, a transmembrane domain, and an extracellular marker domain, wherein the marker domain specifically binds a bispecific polypeptide comprising a marker binding domain comprising a single chain polypeptide and a cell surface protein binding domain comprising a single chain polypeptide that binds a cell surface receptor of a cell, wherein the marker domain contains a structural inert domain from human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19 and EGFR, and wherein the marker binding domain is the sequence set forth in SEQ ID NO: 28; and (iii) a terminator region operable to terminate transcription in an immune cell; and the immune cells comprise a second nucleic acid vector, the second nucleic acid vector comprising (iv) a promoter region operable for transcription in an immune cell; (v) a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules; and (vi) a terminator region operable to terminate transcription in an immune cell; and (b) an effective amount of a bispecific polypeptide to activate the proliferated immune cells to express an immunomodulatory polypeptide, the bispecific polypeptide comprising a cell surface protein binding domain and a marker binding domain having a defined amino acid sequence that specifically binds to a marker domain expressed by the proliferated immune cells, the cell surface protein binding domain specifically binds to a cell surface receptor of a tumor cell, wherein the marker domain contains a structural inert domain from human mesothelin ECD and the cell surface receptor is selected from the group consisting of BCMA, CD19 and EGFR.

71. The use of claim 70, wherein the compound is ###0010### wherein the tumor cell is a mesothelial cell overexpressing mesothelin and PDL1, and the cell surface protein is mesothelin expressed on the surface of the mesothelial cell, and wherein the effector molecule comprises a VHH domain that specifically binds to PD-1 or CD40.

72. The use of claim 70, wherein the compound is ###0017### wherein the tumor cell is a B cell, and the cell surface protein is CD19 on the surface of the B cell.

73. The use of any one of claims 70-72, wherein the antibody is administered at a dose of about 10 mg / kg. wherein the immune cell is a T cell, a TIL cell, a cytokine-activated killer cell, a dendritic cell-cytokine-activated killer cell, a gd-T cell, a NKT cell, or a natural killer cell.

Images