Genetically engineered T cells

Abstract

A therapeutic molecule (single-chain based antibody or ligand based) optimized for expression and secretion from genetically engineered T cells that are gamma delta (γδ) T cells. When expressed from the genetically engineered γδ T cells, the STAR is secreted and mediates binding of the γδ T cell to an antigen / receptor on a target cell. Binding mediates the formation of a cytolytic synapse between the γδ T cell and the target cell, activating the γδ T cell and releasing proteolytic enzymes that kill the target cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 601,999, filed November 22, 2023, and U.S. Provisional Patent Application No. 63 / 428,181, filed January 10, 2023, the entire disclosures of which are incorporated herein by reference.

[0002] Sequence Listing The nucleic acid and amino acid sequences set forth in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file named "240110_103-3002PCT3_Seq_Listing.xmL" (~252kb), created January 10, 2024, which is incorporated herein by reference. Summary of the Invention

[0003] Novel T cell-activating bispecific antibody therapeutics. In some variations, the bispecific antibodies can be used to engage cytotoxic T cells against tumor cells. Genetically engineered gamma delta T cells secrete bispecific therapeutics (antibody- and / or ligand-based) to enhance cytotoxicity to various tumor antigens. [Brief explanation of the drawings]

[0004] [Figure 1] Figure 1 provides an exemplary STAR framework. [Figure 2] FIG. 2 provides a schematic showing the subject matter of variable elements and exemplary specific elements of a general STAR design. [Figure 3]Figure 3 further shows a schematic diagram depicting the variable elements of the STAR design. [Figure 4] FIG. 4 shows a schematic of an alternative STAR design capable of binding to gamma delta T cells. [Figure 5] FIG. 5 is a schematic diagram of LentET STAR. [Figure 6] FIG. 6 provides a schematic diagram of the method for producing gamma delta T cells and the genetically engineered gamma delta T cells. [Figure 7] FIG. 7 is a schematic diagram of the mechanism of action of STARS. [Figure 8] FIG. 8 is a flow diagram of a method for the production of gdT cells. [Figure 9] Figure 9 is an overview of the gdT cell proliferation process. [Figure 10] FIG. 10 shows the identification of donors with acceptable ex vivo expansion of gdT cells from peripheral blood mononuclear cells (PBMCs). [Figure 11] FIG. 11 shows screening of ex vivo expanded gdT cells to identify donors that generate gdT cells with high cytotoxicity against K562 human cancer cells. [Figure 12] FIG. 12 is a chart of GFP expression in LentET-transduced gdT cells. [Figure 13] FIG. 13 is a chart of GFP MFI in LentET-transduced gdT cells. [Figure 14] FIG. 14 is a chart of data showing the cytotoxicity of secreted media from PTK7 and GD2 STAR-expressing 293T cells. [Figure 15] FIG. 15 shows data on the cytotoxicity of mRNA-transfected gdT-CMK. [Figure 16] Figure 16 is a Western blot analysis of designed STAR proteins. [Figure 17] FIG. 17 shows the quantification of STAR secretion. [Figure 18]FIG. 18 is a secretion profile with an albumin linker. [Figure 19] FIG. 19. Characterization of gdT cells transduced with STAR-encoding lentiviruses that potentially acquire cytotoxicity against target cells. [Figure 20] FIG. 20 presents data characterizing Integrin aV B3 CD3 STAR. [Figure 21] Figure 21 presents data characterizing IL2 CD19 CD3 STAR. [Figure 22] FIG. 22 presents data characterizing mSA PTK7 CD3 STAR. [Figure 23] FIG. 23 presents data characterizing mSA PTK7 CD3 STAR. [Figure 24] FIG. 24 presents data characterizing mSA and native signal peptide hSCF CD3 STARs. [Figure 25] Figure 25 shows gdT cells (effector [E]) were transfected with mRNA encoding the mSA and native signal peptide versions of hSCF CD3 STAR and mixed with IMR5 cells (target [T]). [Figure 26] Figure 26 presents data characterizing mSA and IL2 GD2 CD3 STARs. [Figure 27] Figure 27 presents data characterizing mSA and IL2 GD2 CD3 STARs. [Figure 28] Figure 28 shows IL2 SSTR HL and LH CD3 STAR. [Figure 29] Figure 29 shows IL2 SSTR HL and LH CD3 STAR. [Figure 30] FIG. 30 shows that a humanized / deimmunized version of CD3 scFv directs gdT-mediated killing. [Figure 31] FIG. 31 shows that humanized / deimmunized versions of CD3 scFv direct gdT-mediated killing. [Figure 32]FIG. 32 shows lentiviral delivery of shRNA to knock down surface expression of HLA class I and II. [Figure 33] FIG. 33 shows lentiviral delivery of shRNA to knock down surface expression of HLA class I and II. [Figure 34] FIG. 34 shows that alternative gdT-targeting moieties direct gdT-mediated cytotoxicity. [Figure 35] FIG. 35 shows that alternative gdT-targeting moieties direct gdT-mediated cytotoxicity. [Figure 36] FIG. 36 shows somatostatin ligand gdT-mediated cytotoxicity to NET cells. [Figure 37] FIG. 37 shows somatostatin ligand gdT-mediated cytotoxicity to NET cells. [Figure 38] Figure 38 shows IL2 TPO BR CD3 STAR expression. [Figure 39] FIG. 39 shows mRNA-mediated protein expression correlated with mRNA free energy. [Figure 40] FIG. 40 shows flow cytometry data comparing the killing activity of PTK7-14, SSTR2-3, and SSTR2-8, representing percent killing activity at 1:1 and 5:1 ratios. [Figure 41] Figure 41 is a Western blot showing that the desired protein was expressed from the plasmid DNA. [Figure 42] FIG. 42 shows flow cytometry data showing percent killing of target cells comparing untreated vs. GD2-3. [Figure 43] Figure 43 shows the results of STAR-specific IMR5 killing (7AAD+) of transiently transfected 293T cells quantified by ELISA. Cytotoxicity was determined by flow cytometry. [Figure 44]Figure 44 shows the results of STAR-specific IMR5 killing (7AAD+ and / or Annexin V+) of transiently transfected 293T cells quantified by ELISA. Cytotoxicity was determined by flow cytometry. [Figure 45] Figure 45 shows the cytotoxicity of various PTK7-STAR proteins (x-axis) at a 1:1 ratio of effector (gamma delta T cells) to target (IMR5 cells). [Figure 46] Figure 46 shows the cytotoxicity of various PTK7-STAR proteins (x-axis) at a 5:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. [Figure 47] Figure 47 shows the cytotoxicity of various SSTR-STAR proteins (x-axis) at a 1:1 ratio of effector (gamma delta T cells) to target (IMR5 cells). [Figure 48] Figure 48 shows the cytotoxicity of various SSTR-STAR proteins (x-axis) at a 5:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. [Figure 49] Figure 49 shows the cytotoxicity of additional SSTR-STAR proteins (x-axis) at effector (gamma delta T cells) to target (IMR5 cells) ratios of 1:1 and 5:1. Detailed Description of the Invention

[0005] The claimed subject matter will now be described with reference to the drawings, wherein like reference numerals are generally used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the claimed subject matter. It will be apparent, however, that the claimed subject matter may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form in order to facilitate description of the claimed subject matter.

[0006] The drawings and the following description of various systems, methods, and apparatus are not intended to limit the inventive systems, methods, and apparatus disclosed herein to any one variation, but rather to enable anyone skilled in the art of project management and / or software development to make and use the inventive systems, methods, and apparatus.

[0007] The present disclosure relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy. In one variation, the present disclosure discloses hematopoietic cells capable of secreting one or more synthetic fusion proteins and / or therapeutic agents. In particular, the present disclosure relates to the immunotherapy of cancer, including, for example, B-cell malignancies, neuroblastoma, osteosarcoma, neuroendocrine tumors (NETs), and acute myeloid leukemia (AML). In addition, the present disclosure relates to target cell cytotoxic and secretory T cell actuators (referred to herein as "STARs").

[0008] "STARs" is a generic term that refers to proteins genetically engineered to be expressed by gamma delta T cells. The disclosed STARs offer unique advantages over existing soluble immuno-oncology therapies, such as cytokines, monoclonal antibodies, and bispecific immune cell engagers. The STARs disclosed herein provide solutions to side effects (i.e., dose, pharmacokinetic, and pharmacodynamic) encountered with existing soluble immuno-oncology therapies. The STARs disclosed herein are ECO-optimized using a proprietary codon optimization method. STARs are novel ECO-optimized secretory T cell actuators that are secreted from gamma delta T cells via gene transfer (e.g., viral vector transduction or mRNA electroporation).

[0009] [Means for solving the problem] Disclosed herein are novel gamma delta T cells (referred to herein as gdT cells or γδT cells) engineered to secrete proteins that can affect cancer. In one example, the gdT cells are engineered to secrete proteins that act to alter the growth, proliferation, and viability of a T cell population. In one variation, the gamma delta T cells secrete bispecific T cell actuators. Traditionally, bispecific T cell engagers have been injected directly into patients via bolus therapy of Fc-containing bispecific antibodies (bsAbs) or continuous infusion of Fc-free bsAbs. Disclosed herein are novel methods for introducing STARs, T cell actuators, and / or other bispecific molecules and / or other secreted proteins by delivering gdT cells capable of secreting a therapeutic agent of interest to a patient. In some variations, the gdT cells delivered to a patient are capable of secreting a protein of interest. In one variation, gdT cells expressing STARs, e.g., STARs (e.g., bispecific T cell engagers), are introduced into the patient.

[0010] This disclosure encompasses STAR designs that target gdT cells to somatostatin receptor 2 (SSTR2)+ tumor cells. SSTR2-targeting monoclonal antibodies were adapted by converting them into several single-chain variable fragment (scFv) designs, which were used to direct STARs / gdT cells to SSTR2+ tumors.

[0011] In the present disclosure, the STAR designs can be secreted in vivo from ex vivo modified gdT cells. However, the protein designs can also be utilized as directly injected recombinant proteins. The gdT cells can be modified to express the STARs by a number of methods, including lentiviral transduction, AAV transduction, mRNA electroporation, mRNA transfection, and non-viral gene transfer techniques, such as CRISPR knock-in.

[0012] Codon optimization is a genetic engineering approach to improving gene expression by altering synonymous codons based on the codon bias of an organism. Disclosed herein are expression codon optimization (ECO) sequences for amino acid sequences, non-optimized DNA sequences, and gamma delta T cell (ECOg) sequences. Expression codon optimization for gamma delta T cell expression uses an algorithm with novel codon usage indicators generated from expression data of the target cell, in this case, gamma delta T cells. When sequences are optimized for expression in or by gamma delta T cells, they may be interchangeably referred to as ECOg or gamma delta T cell-optimized sequences. This means that the sequences have been codon-optimized to improve expression from gamma delta T cells compared to wild-type or non-optimized sequences. Additionally, two unique codon-optimized sequences for the IL2 signal peptide are disclosed that are uniquely ECO-optimized to enhance translation initiation for improved protein expressibility. The IL2 signal peptide was optimized using a unique method in conjunction with the optimization of the STAR scFv-containing domain. Furthermore, these sequences in the LentET lentiviral backbone of the present disclosure use two different promoters active in gdT cells (see Figure 5). The first is the synthetic MND promoter (SEQ ID NO: 152); the second is the heat shock 70 kDa protein 8 promoter HSPA8 (SEQ ID NO: 153) derived from the human genome. The latter promoter was found to have high activity in gamma delta T cells. The use of this promoter to drive gene expression in gamma delta T cells, particularly from a lentivector, is a novel use of this sequence.

[0013] Other systems, methods, features, and advantages of the present disclosure will be, or will become, apparent to one with skill in the art upon examination of the figures and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this specification, be within the scope of the disclosure, and be protected by the following claims.

[0014] [Effects of the invention] Disclosed herein are T cells that eradicate cancer cells through various pathways, for example, as a result of secreting therapeutic agents such as STARs. A further advantage of this method is that the disclosed STARs can also recruit the patient's T cells to fight cancer.

[0015] Additional or alternative techniques include, but are not limited to, the addition of molecules that enhance T cell function, e.g., gamma delta T cell function. Molecules that improve T cell proliferation and survival in vivo can also be added. Some examples of additional molecules are IL-2 and IL-15. In some embodiments, the STAR is a bispecific T cell actuator. In other variations, the STAR operates without engaging the T cell with cancer cells. In some variations, the STAR mediates T cell proliferation.

[0016] Disclosed herein is a STAR, a protein secreted by γδ T cells, with unique properties. Secretion from γδ T cells has not previously been demonstrated. Indeed, secretion from γδ T cells required extensive optimization of the expression construct. To achieve the disclosed constructs expressible from γδ T cells, the system was optimized at multiple points in the protein expression pathway, as described below.

[0017] STARs (e.g., single-chain-based antibody and / or ligand-based) are optimized for expression and secretion from genetically engineered gamma delta ("(γδ) T cells" or "gdT"). When a STAR is expressed from the genetically engineered γδ T cell, the STAR is secreted and mediates binding of the γδ T cell to an antigen / receptor on a target cell. Binding mediates the formation of a cytolytic synapse between the γδ T cell and the target cell, activating the γδ T cell and releasing proteolytic enzymes that kill the target cell.

[0018] Disclosed herein are STARs (e.g., scFv-based antibody and / or ligand-based formats) that are optimized for gdT cell expression and secretion (IL-2 signal peptide sequence or another signal peptide).

[0019] term Unless otherwise specified, technical terms are used according to conventional usage.The definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishing House, 1995 (ISBN 1-56081-569-8).

[0020] To facilitate understanding of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0021] Adeno-associated virus (AAV): A small, replication-deficient, non-enveloped virus that infects humans and other primates. AAV is not known to cause disease and elicits only a very mild immune response. AAV-based gene therapy vectors can infect both dividing and quiescent cells and persist extrachromosomally without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. Currently, 11 AAV serotypes (AAV1-11) are recognized.

[0022] Administer / Administer: To provide or administer an agent, such as a therapeutic agent (e.g., a recombinant AAV, a recombinant lentivirus, a STAR, a vector expressing a STAR, a modified gdT cell capable of expressing a STAR), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, tube, sublingual, rectal, transdermal, nasal, vaginal, and inhalation.

[0023] Antigen-binding site: As used herein, the term "antigen-binding site" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding site can guide its bound entity (e.g., a second antigen-binding site) to a target moiety, such as a specific type of tumor cell bearing the antigenic determinant. In another embodiment, an antigen-binding site can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding sites include antibodies and fragments thereof, as further defined herein. Particular antigen-binding sites include the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In one embodiment, an antigen-binding site includes an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0024] Antigenic determinant: As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a stretch of contiguous amino acids or a three-dimensional structure composed of different stretches of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding site binds to form an antigen-binding site-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0025] Specific binding: "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigenic determinant can be measured using either an enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art. In one embodiment, the extent of binding of the antigen-binding site to an unrelated protein is less than about 10% of the binding of the antigen-binding site to the antigen as measured, for example, by SPR. In certain embodiments, an antigen-binding site that binds to an antigen, or an antibody comprising that antigen-binding site, exhibits a dissociation constant (K D ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 (up to M).

[0026] Affinity: "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding site and an antigen, or a receptor and its ligand). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K D ), which can be expressed as the dissociation rate constant and the association rate constant (k off and k on ) ratio. Thus, different rate constants can result in comparable affinities as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods well known in the art, including those described herein.

[0027] The terms "first," "second," or "third," as used herein with respect to Fab molecules etc., are used for convenience to distinguish between sites of each type when multiple sites are present. The use of these terms is not intended to confer a particular order or orientation of the bispecific antibody unless explicitly stated.

[0028] Valence: As used herein, the term "valence" refers to the presence of a specific number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" refers to the presence of one (or at most one) antigen-binding site in the antibody that is specific for that antigen.

[0029] Antibody: As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0030] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0031] Antibody fragment: "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), and single-domain antibody. Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0032] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain involved in binding between the antibody and an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0033] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0034] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are also called α, δ, ε, γ, and μ, respectively.

[0035] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of an immunoglobulin light chain (a "Fab light chain").

[0036] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced with each other); i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH1, NC-terminal direction) and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, NC-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0037] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its natural form, i.e., a Fab molecule comprising a heavy chain made up of heavy chain variable and constant domains (VH-CH1, N- to C-terminal), and a light chain made up of light chain variable and constant domains (VL-CL, N- to C-terminal).

[0038] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 Da, consisting of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain contains a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain contains a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains are classified into five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can be classified into one of two types, also called κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and one Fc domain, connected via an immunoglobulin hinge region.

[0039] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term encompasses native-sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is usually defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain or a truncated variant of the full-length heavy chain, in which case the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present.

[0040] "Decreased binding," e.g., decreased binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured, for example, by SPR. For clarity, this term also encompasses a decrease in affinity to zero (or below the detection limit of the method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0041] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0042] Gamma delta T cells (γδ T cells) or gdT cells are T cells that have a distinctive T cell receptor (TCR) on their surface. Most T cells are αβ (alpha beta) T cells, whose TCR consists of two glycoprotein chains, also called the α (alpha) TCR chain and the β (beta) TCR chain. In contrast, γδ (gamma delta) T cells have a TCR consisting of one γ (gamma) chain and one δ (delta) chain. This T cell population is usually less frequent than αβ T cells.

[0043] Hematopoietic cells are cells that can develop into blood cells through hematopoiesis.

[0044] Human peripheral blood mononuclear cells (PBMCs) are immune cells with a single nucleus. PBMCs are derived from the bone marrow. PBMCs are secreted into the peripheral circulation. PBMCs are involved in both humoral and cell-mediated immunity. PBMCs include lymphocytes (T cells, B cells, and NK cells) and monocytes.

[0045] Unless otherwise specified, "CD3" refers to any native CD3 from a vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term "full-length" encompasses unprocessed CD3 and all forms of CD3 generated by intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3c). The amino acid sequence of human CD3epsi is set forth in UniProt (www.uniprot.org) accession no. P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1.

[0046] The BiTE format, also known as tandem scFv or (scFv)2, is a small, Fc-free molecule in which two scFvs are linked on a single polypeptide by a flexible linker. In vivo delivery of the genetic information encoding the bsAb can be achieved using viral or non-viral vectors.

[0047] Bispecific: The term "bispecific" (including bi-specific and bsAb) means that an antibody can specifically bind to at least two different antigenic determinants. Typically, a bispecific antibody contains two antigen-binding sites, each specific for a different antigenic determinant. In certain embodiments, a bispecific antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0048] Bispecific antibodies comprise at least one or more antigen-binding domains; a multimerization core that forms homo- or heteromultimers; and a linker connecting the elements. The antigen-binding domains can be antibody fragments, such as Fabs, single-chain variable fragments (scFvs), or single-chain domain antibodies (sdAbs), or antibody mimetics. Another approach is to use the extracellular domains of natural receptors or ligands in bsAbs design. The multitargeting concept enabled by bsAbs is particularly attractive from a therapeutic perspective because diseases are multifactorial and involve multiple receptors, ligands, and signaling cascades. T cell engager bsAbs (TCEs) are designed to simultaneously bind selected tumor-associated antigens (TAAs) on the surface of tumor cells and one extracellular CD3 subunit (most commonly CD3e) on the surface of T cells.

[0049] cDNA (complementary DNA): A fragment of DNA lacking internal non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. cDNA may also contain untranslated regions (UTRs) that are responsible for translational control of the corresponding RNA molecule.

[0050] Codon optimization: A "codon-optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been changed to optimize expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species (such as human cells). Codon optimization does not change the amino acid sequence of the encoded protein.

[0051] CAI: "CAI" is the Codon Adaptation Index. CAI is used as a quantitative method to predict the expression level of a gene from its codon sequence.

[0052] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with cancer. In still other embodiments, the control is a historical control or standard reference value or range of values ​​(such as a previously tested control sample, such as a group of cancer patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

[0053] The difference between the test sample and the control sample can be an increase or a decrease. The difference can be qualitative or quantitative, e.g., a statistically significant difference. In some embodiments, the difference is an increase or decrease of at least about 5%, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% relative to the control.

[0054] DNA (deoxyribonucleic acid): DNA is the long-chain polymer that contains the genetic material of most living organisms (although some viruses have genes containing ribonucleic acid (RNA)). The repeating units of a DNA polymer are four different nucleotides, each containing one of four bases—adenine (A), guanine (G), cytosine (C), and thymine (T)—joined to a deoxyribose sugar with a phosphate group attached. A triplet of nucleotides (also called a codon) codes for each amino acid in a polypeptide or a stop signal. The term codon is also used for the corresponding (and complementary) sequence of three nucleotides in the mRNA into which the DNA sequence is transcribed.

[0055] Unless otherwise specified, a reference to a DNA molecule is intended to encompass the reverse complement of that DNA molecule.Unless otherwise specified, a DNA molecule is required to be single-stranded herein, even if only a single strand is described, it encompasses both strands of a double-stranded DNA molecule.Therefore, a reference to a nucleic acid molecule that encodes a specific protein or a fragment thereof encompasses both the sense strand and its reverse complement.For example, it is appropriate to prepare a probe or primer from the reverse complement sequence of the disclosed nucleic acid molecule.

[0056] Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

[0057] Flanking: Near or adjacent, including further adjacent, in a linear or circular polynucleotide such as a DNA molecule.

[0058] Gene: A nucleic acid sequence, typically a DNA sequence, that includes regulatory and coding sequences necessary for transcription of an RNA, whether or not it is an mRNA. For example, a gene may include a promoter, one or more enhancers or silencers, a nucleic acid sequence encoding an RNA and / or polypeptide, downstream regulatory sequences, and possibly other nucleic acid sequences involved in regulating the expression of an mRNA.

[0059] As is well known in the art, most eukaryotic genes contain both exons and introns. The term "exon" refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and / or experimentally confirmed to contribute to the contiguous sequence of a mature mRNA transcript. The term "intron" refers to a nucleic acid sequence found in genomic DNA that is predicted and / or confirmed not to contribute to the mature mRNA transcript, but rather is "spliced ​​out" during processing of the transcript.

[0060] Gene therapy: The introduction of a heterologous nucleic acid molecule into one or more recipient cells, where expression of the heterologous nucleic acid in the recipient cells affects the function of the cells and provides a therapeutic effect to the subject. For example, the heterologous nucleic acid molecule can encode a protein that affects the function of the recipient cells.

[0061] Hybridization: Hybridization assays for characterizing nucleic acids that have a certain level of homology with the nucleic acid sequences provided herein are well known in the art; see, for example, Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989). As used herein, the term "hybridization" or "hybridize" can refer to hybridization under stringent or non-stringent conditions. Unless further specified, the conditions are preferably non-stringent. The hybridization conditions can be established according to conventional protocols, for example, as described in Sambrook (2001) loc.cit.; Ausubel (1989) loc.cit., or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach", IRL Press, Oxford, Washington DC (1985). Setting conditions is within the skill of those skilled in the art and can be determined according to protocols described in the art. Thus, to detect only specifically hybridized sequences, stringent hybridization and washing conditions are usually required, such as highly stringent hybridization at 65°C, 0.1xSSC, 0.1% SDS, or 2xSSC, 60°C, 0.1% SDS. Low stringency hybridization conditions for detecting homologous sequences or sequences that are not exactly complementary may be set, for example, at 6xSSC, 1% SDS, and 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

[0062] Intron: a stretch of DNA within a gene that does not contain protein-coding information. Introns are removed before the messenger RNA is translated.

[0063] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at both ends of the AAV DNA genome. ITRs function as origins of replication for viral DNA synthesis and are essential cis-components for the generation of AAV integrative vectors.

[0064] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) is one that has been substantially separated or purified from the cells or tissues of the organism in which it naturally occurs, or from other biological components of the organism itself, such as other chromosomal and extrachromosomal DNA, and RNA, proteins, and cells. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0065] Nucleic acid molecule: A polymer of nucleotides, including both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic and mixed polymers thereof. Nucleotide refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. As used herein, the term "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." Nucleic acid molecules are typically at least 10 bases in length, unless otherwise specified. The term encompasses single- and double-stranded DNA. Polynucleotides can include either naturally occurring nucleotides and modified nucleotides linked by linkages with naturally occurring and / or non-naturally occurring nucleotides. "cDNA" refers to DNA complementary to or identical to mRNA, in single- or double-stranded form. "Encode" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom.

[0066] Nucleotide: This term includes, but is not limited to, monomers that contain a base linked to a sugar, such as pyrimidine, purine, or their synthetic analogs, or a base linked to an amino acid, such as peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. Nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0067] Operable linkage: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, in the same reading frame.

[0068] ORF (open reading frame): A series of nucleotide triplicates (codons) that code for amino acids. These sequences are usually translatable into peptides.

[0069] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers used are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed vectors.

[0070] Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition (e.g., vector composition) to be administered can contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, e.g., sodium acetate or sorbitan monolaurate, preservatives, and pH buffering agents. In a specific embodiment, carriers suitable for administration to a subject can be sterile and / or can be suspended or otherwise contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing the desired immune response. They can also be accompanied by pharmaceutical agents for use in therapeutic purposes. The unit dosage form may be, for example, in a sealed vial containing sterile contents, or in a syringe for injection into a subject, or in a lyophilized form for subsequent solubilization and administration, or in a solid or controlled release form.

[0071] Polypeptide: A chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are non-natural amino acids, e.g., artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into the polypeptide by an amide bond or amide bond mimetic. Polypeptides have an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and refers herein to a polymer of amino acid residues.

[0072] Prevention, Treatment, or Amelioration of Disease: "Prevention" of a disease (such as cancer) refers to inhibiting the full development of the disease. "Treatment" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has begun to develop. "Amelioration" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0073] Promoter: A region of DNA that directs / initiat- es the transcription of a nucleic acid (e.g., a gene). A promoter includes necessary nucleic acid sequences near the transcription start site. Typically, promoters are located near the gene they transcribe. A promoter also optionally includes distal enhancer or repressor elements, which may be located as much as several thousand base pairs away from the transcription start site. A tissue-specific promoter is a promoter that directs / initiat- es transcription primarily in one type of tissue or cell.

[0074] Protein: A biological molecule that is expressed by a gene or other encoding nucleic acid (e.g., cDNA) and contains amino acids.

[0075] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been separated, in whole or in part, from naturally occurring associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

[0076] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not found in nature, e.g., a sequence that includes one or more nucleic acid substitutions, deletions, or insertions, and / or a sequence that is made by the artificial combination of two otherwise separate sequence segments, which can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid segments, e.g., by genetic engineering techniques.

[0077] A recombinant virus is one that comprises a genome that includes a recombinant nucleic acid molecule. As used herein, "recombinant AAV" refers to an AAV particle into which a recombinant nucleic acid molecule is packaged.

[0078] A recombinant protein has a sequence that does not occur in nature or that is created by the artificial combination of two separate sequence segments. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial cell or a eukaryotic cell, or into the genome of a recombinant virus.

[0079] Response element (RE): A DNA sequence, including a promoter, to which one or more transcription factors can bind and provide an aspect of control over gene expression.

[0080] Sequence identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more identical the sequences. Sequence similarity can be measured in terms of percent similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess relatively high sequence identity / similarity when aligned using standard methods. This homology is more pronounced when orthologous proteins or cDNAs are derived from more closely related species (such as human and mouse sequences) compared to more distantly related species (such as human and nematode sequences).

[0081] Additionally or alternatively, percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent identity, and not considering some conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of a person in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. Amino acid identity is provided in the output alignment header.

[0082] This method of aligning sequences for comparison is known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgin & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al. al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, which present a detailed discussion of sequence alignment methods and homology calculations.

[0083] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. See the NCBI website for additional information.

[0084] As used herein, a reference to "at least 90% identity" refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a particular reference sequence.

[0085] Subject: Vertebrate multicellular organisms, a category that includes both human and non-human mammals.

[0086] Synthetic: Produced by artificial means in a laboratory, for example, synthetic nucleic acids can be chemically synthesized in a laboratory.

[0087] TATA box: a DNA sequence found in the promoter region of a gene that allows TATA binding protein and transcription factor IID to bind when the DNA unwinds and RNA polymerase II binds. TATA box sequences typically contain the sequence TATAAA and often contain an additional 3' adenine nucleotide.

[0088] Therapeutically effective amount: An amount of a particular pharmaceutical or therapeutic agent (e.g., recombinant AAV) sufficient to achieve a desired effect in a subject or cell treated with the agent. The effective amount of an agent depends on several factors, including, but not limited to, the subject or cell being treated and the method of administration of the therapeutic composition.

[0089] Transcription factor (TF): A protein that binds to specific DNA sequences and thereby controls the transfer (or transcription) of genetic information from DNA to RNA. TFs perform this function alone or in complexes with other proteins by promoting (as activators) or inhibiting (as repressors) the recruitment of RNA polymerase (the enzyme that transcribes genetic information from DNA to RNA) to specific genes. The specific DNA sequences to which TFs bind are known as response elements (REs) or control elements. Other names include cis-elements and cis-acting transcriptional regulatory elements.

[0090] Transcription factors interact with their binding sites using a combination of electrostatic forces (hydrogen bonds are a special case) and van der Waals forces. Due to the nature of these chemical interactions, most transcription factors bind to DNA in a sequence-specific manner. However, not all bases in a transcription factor binding site actually interact with the transcription factor. Furthermore, some of these interactions may be weaker than others. Thus, many transcription factors may not bind to a single base sequence, but rather to a subset of closely related base sequences, each with varying interaction strengths.

[0091] For example, the consensus binding site for TATA binding protein (TBP) is TATAAAA, but the TBP transcription factor can also bind to similar sequences such as TATATAT or TATATAA.

[0092] Transcription factors (TFs) are classified based on many aspects, such as secondary, tertiary, and quaternary protein structure, DNA binding sequences and properties, interactions with the DNA double helix, metal and other binding characteristics, etc. The JASPAR database and TRANSFAC (TRANSFAC® 7.0 Public 2005) are two web-based transcription factor databases, their experimentally proven binding sites, and the genes they regulate.

[0093] Transcription start site: The location at the 5' end of a gene sequence where transcription begins.

[0094] Therapeutically effective amount: An amount of an agent, such as a recombinant AAV vector, sufficient to prevent, treat (including prophylactic), reduce, and / or ameliorate the symptoms and / or underlying causes of a disorder or disease, e.g., to prevent, inhibit, and / or treat cancer. For example, this can be the amount necessary to inhibit or prevent viral replication or to measurably alter the outward symptoms of a disease or condition.

[0095] For example, administration of a therapeutically effective amount of a vector disclosed herein can reduce symptoms by a desired amount, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100% or more, compared to a suitable control.

[0096] It is understood that in order to achieve a therapeutic response to disease or pathological condition, the drug needs to be administered multiple times.Thus, a therapeutically effective amount includes a divided amount that contributes to achieve a therapeutic result in patients in combination with previous or subsequent administrations.For example, a therapeutically effective amount of a drug can be administered once, or can be administered several times during a treatment period, for example, daily.However, a therapeutically effective amount can depend on the subject being treated, the severity and type of the pathological condition being treated, and the method of administration.The unit dosage form of a drug can be packaged in a single treatment amount or multiple treatment amounts, for example, in a vial (e.g., with a pierceable cap) or syringe with sterilized components.

[0097] Vector: A vector is a nucleic acid molecule that allows the insertion of a foreign nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector can include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of the inserted gene or genes. In some embodiments herein, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a gammaretrovirus vector, a lentivirus vector, or an adenovirus vector.

[0098] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. "Comprising A or B" means including A, B, or A and B. Furthermore, it is understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials and methods, as well as the examples, are illustrative and not intended to be limiting.

[0099] Summary of the Invention According to the present disclosure, the term STARs refers to secretory T cell actuators ( s ecreted T cell A ctuato r s). The term "actuator" is used to encompass T cell engagers and / or activators. The term "actuator" refers to a secreted molecule that enables gamma delta T cells to perform a therapeutic function. STARs may be composed of a STAR (scFv-based antibody or ligand-based) on one end and a T cell actuator molecule (scFv-based antibody or ligand-based) on the other end. Each component of the STAR construct may have a sequence optimized for expression and secretion by gamma delta T cells.

[0100] In one variation, STARs are bispecific and / or biactive therapeutic molecules secreted from genetically engineered γδ T cells. The STARs mediate binding of CD3 on the γδ T cell to a ligand molecule on the target cell, forming an immunological synapse and activating the cytotoxicity of the γδ T cell.

[0101] Figure 1 provides a schematic of an exemplary STAR framework. A STAR may include at least one of the following elements: starting from the amino terminus and moving to the carboxy terminus, a STAR may include: (a) a signal peptide, which may be cleaved before secretion from the cell, (b) a tumor cell surface protein conjugate (examples are provided below), (c) a flexible linker (optional) (also referred to herein as a "central linker"), (d) a T cell surface protein conjugate.

[0102] Examples of signal peptides according to the present disclosure include, but are not limited to, IL2, mSA (modified serum albumin) (SEQ ID NO: 3) (SEQ ID NO: 5), and hSCF (human stem cell factor) (SEQ ID NO: 4) (SEQ ID NO: 6).

[0103] In one variation, the tumor cell surface binding protein may be, for example, one or more of scFv, Fab, or natural cell ligand. The tumor cell surface binding protein may target, for example, the following cancer cell surface protein targets: CD19, SSTR2, GD2, PTK7, CD5, CD20, CD22, CD110, CD117, CD19 LH scFv, PTK7 HL scFv, GD2 HL scFv, GD2 LH scFv, Integrin aVB3 HL ScFv, SSTR2 HL scFv, SSTR2 LH scFv, 2xSST28 3xG4S, 2xSST28 4xG2s, TPO ligand, hSC ligand (SEQ ID NO: 4). (Note: HL indicates heavy chain-light chain orientation, and LH indicates light chain-heavy chain orientation.) The tumor cell-binding domain may be a single-chain antibody variable domain fragment or tumor cell receptor ligand that binds to one selected from the group consisting of SSTR2, PTK7, GD2, SSTR5, CD19, aVB3, CD110, and CD5. The tumor cell-binding domain may be SSTR2 scFv (SEQ ID NOS: 52-58) (either LH or HL), PTK7 HL scFv (SEQ ID NOS: 17-23), SSTR2 HL scFv, CD19 scFv (SEQ ID NOS: 7-11) (LH or HL orientation), GD2 scFv (SEQ ID NOS: 38-44) (in LH or HL orientation), Integrin aVB3 scFv (SEQ ID NOS: 45-51) (in LH or HL orientation), 2×SST28 It may also be 3×G4S (SEQ ID NOs: 59 to 65), 2×SST28, 4×G2s (SEQ ID NOs: 66 to 72), TPO (ligand) (SEQ ID NOs: 80 to 86), and SCF (ligand) (SEQ ID NOs: 87 to 93), SSTR2-8 (SEQ ID NOs: 156 to 160), GD2-3 (SEQ ID NOs: 161 to 165), or PTK7-14 (SEQ ID NOs: 166 to 170).

[0104] In one variation, the flexible linker may be, for example, G4S (SEQ ID NO: 155), albumin (SEQ ID NOs: 31-37), or Fc (SEQ ID NOs: 87-93). As mentioned herein, the linker may be a combination, for example, G4S refers to the amino acid sequence (GGGGS) (SEQ ID NO: 155), such as G4S-albumin-G4S (e.g., (based on the amino acid sequence) SEQ ID NO: 155-SEQ ID NO: 31-SEQ ID NO: 155); G4S-Fc-G4S (e.g., based on the amino acid sequence) SEQ ID NO: 155-SEQ ID NO: 87-SEQ ID NO: 155).

[0105] In one variation, the gamma delta T cell surface protein binding domain may be, for example, one or more of an scFV, Fab, and / or a natural gamma delta T cell ligand. In one variation, examples of targets of the gamma delta T cell surface antigen proteins disclosed herein include, for example, CD3 subunit, TCR (T cell receptor) subunit, CD16, NKG2D, FasL, and TRAIL. The gamma delta T cell surface antigen protein actuator, engager, and / or activator may be at least one of CD3 HL scFv, CD3(Hum2)HL scFv, JAML HL scFv, CDXAR ligand, gd-c(V1)HL scFv, and gd-c(V6)HL scFv.

[0106] FIG. 2 provides a schematic showing the subject matter of variable elements and exemplary specific elements of a general STAR design.

[0107] Figure 3 shows a further schematic diagram representing variable elements of a STAR design. In the present disclosure, the N-terminal versus C-terminal arrangement of elements is arbitrary. For example, an exemplary CD19 / CD3 molecule may have the CD19 scFV positioned at / towards the N-terminus and the CD3 positioned at / towards the C-terminus. However, it is also possible for CD3 to be positioned at the N-terminus and CD19 at the C-terminus. It is assumed that the IL2 signal sequence (or its surrogate) is always present at the N-terminus. This general scheme can be applied to all molecules described in this disclosure. Additionally, the protein sequence may be reversed relative to its position within the protein.

[0108] Figure 4 shows a schematic of an alternative STAR design capable of binding gamma delta T cells. A ligand-based STAR is shown with cytokine actuators at both ends of the construct. In this exemplary STAR, the signal peptide can be, for example, IL2, mSA (SEQ ID NO: 3), or hSCF (SEQ ID NO: 4). Cytokine 1 can be IL2 or IL15. The optional linker (e.g., a flexible linker or central linker) can be albumin (e.g., SEQ ID NOs: 31-37) or Fc (SEQ ID NOs: 87-93), and cytokine 2 can be IL15 or IL2.

[0109] Figure 5 is a schematic diagram of a LentET STAR. It shows elements that can be variably present in the transgene design on a lentiviral cassette for expressing a STAR from target cells. The transgene can include one or more of an shRNA cassette (optionally), an internal promoter (e.g., MND or HSPA8), and a WPRE sequence (optionally). LentET is a lentiviral packaging system. In one variation, the transgene packaging plasmid is driven by an external CMV promoter that drives expression of a transgene cassette RNA that includes one or more of the following: 1) cis-viral elements necessary for virus assembly and packaging, 2) an internal promoter sufficient for gene expression in the target tissue (e.g., the MND promoter (SEQ ID NO: 152) or the HSPA8 promoter (SEQ ID NO: 153), 3) an ECO-optimized STAR-encoding cDNA sequence, 4) optionally a mutated form of the woodchuck hepatitis virus post-transcriptional regulator (WPREmut), and 5) optionally an shRNA expression cassette driving expression of a directed shRNA against a component of the major histocompatibility class (MHC) I or II complex.

[0110] Figure 6 provides a schematic diagram of methods for producing and genetically engineering gamma delta T cells. Autologous or allogeneic donor peripheral blood mononuclear cells (PBMCs), which can be pre-screened for disease-specific profiles, can be used as starting material for the expansion, genetic engineering, and purification of genetically modified gamma delta T cells. The final product can include various compositions of gamma delta T cells, which can include, for example, various ratios of gamma delta 1+ and gamma delta 2+ T cells, for adoptive transfer and treatment of various hematological cancers and solid tumors.

[0111] Also disclosed are gamma delta T cell methods and compositions, which may include gamma delta 1+ and gamma delta 2+ T cells genetically engineered to express and / or secrete therapeutic agents against tumor antigens, for example, for the treatment of various hematological cancers and solid tumors. Gamma delta T cells can be expanded under serum-free conditions from autologous or allogeneic donors. Donors can be selected based on a range of screening criteria, including, but not limited to, disease-specific / target-specific profiles, such as cytotoxicity assays in the presence or absence of other drugs and / or immunotherapies. Gamma delta T cell expansion can be performed under serum-free conditions in a two-phase expansion procedure using autologous or allogeneic PBMCs as starting material. Briefly, in one variation, PBMCs are divided into two cultures: 1) expansion of gamma delta 2+ T cells in the presence of zoledronic acid and IL-2, and 2) expansion of gamma delta 1+ T cells by activation with a delta 1 monoclonal antibody in the presence of IL-2. Both expansion procedures can involve two phases: the first in the presence of the indicated supplements followed by depletion of αβ T cells; and the second in the presence of IL-2 alone. Alternatively, gamma delta 1+ T cells can be expanded by exposure to concanavalin A (Con A) or phytohemagglutinin (PHA) stimulation in the presence of IL-2.

[0112] Genetic modification can occur during the first or second phase of growth. Modification in the first phase can be achieved using a modality that genetically modifies chromosomal DNA, for example, using viral or non-viral approaches. These approaches include, but are not limited to, lentivirus, gammaretrovirus, CRISPR, TALEN, etc. Genome modification in this phase can be inherited by all daughter cells produced during further growth. Modification in the second phase can be achieved using non-integrative approaches that are not inherited by daughter cells, such as AAV or mRNA.

[0113] In this disclosure, STARs can be secreted from genetically engineered gamma delta T cells. Actuation can occur through several mechanisms, occurring alone or in combination. STARs mediate binding of CD3 (or other T cell ligands) on gamma delta T cells to ligand molecules on target cells, forming an immunological synapse and activating T cell cytotoxicity. Secreted STARs also mediate binding of non-genetically engineered gamma delta T cells to ligand molecules on target cells, forming an immunological synapse and activating T cell cytotoxicity.

[0114] In the present disclosure, genetic modifications can be performed in gamma delta 1+ T cells and gamma delta 2+ T cells in combination with or separately with STARs and / or chimeric antigen receptors (CARs).

[0115] Figure 7 is a schematic diagram of the mechanism of action of STARS. This mechanism of action may involve the expression and secretion of STARs from genetically engineered T cells, which mediate binding of T cells to antigens / receptors on target cells. Formation of a cytolytic synapse between the T cell and target cell by STARs results in the release of proteolytic enzymes that mediate T cell activation and target cell cytotoxicity. Secretion of STARs from genetically engineered T cells also leads to binding of unmodified T cells to target cells, resulting in enhanced cytotoxicity. Genetic engineering of T cells with STARs can be combined with other immunotherapeutic approaches, such as, but not limited to, chimeric antigen receptors (CARs), monoclonal antibodies, and / or cytotoxicity-enhancing molecules. Molecules of the invention can also be produced and purified from in vitro expression systems and delivered as recombinant protein products.

[0116] By preparing final formulations with varying ratios of gamma delta 1+ / gamma delta 2+ T cells, tumor-specific targeting and enhanced cytotoxicity are mediated based on the unique characteristics of each gamma delta T cell type, as summarized in Table 1:

[0117] [Table 1]

[0118] In aspects of the present disclosure, gdT cell codon-optimized scFv and ligand nucleic acid sequences are provided as SEQ ID NOs: 1-185. In this disclosure, T cells can be genetically engineered via plasmids, mRNA, AAV, lentivirus, or retrovirus. According to this disclosure, the genetically engineered T cells can provide endogenous expression and secretion of STARs for enhanced cytotoxicity and can be used for adoptive cell transfer.

[0119] According to the present disclosure, production of recombinant STARs can be used as therapeutic agents for cancer and solid tumors, including neuroendocrine tumors (NETs) and neuroblastoma. Therapeutic mechanisms may include sequence-optimized expression and secretion of STARs from genetically engineered T cells for autocrine / paracrine binding between T cells and target tumor cells.

[0120] In accordance with the present disclosure, the STARs may be recombinant protein products or recombinant purified molecules for direct use as therapeutic agents. The present disclosure encompasses endogenous expression of STARs and / or adoptive cell transfer of T cells, which may enhance the cytotoxic function of not only the adoptively transferred cells but also endogenous T cells.

[0121] According to the present disclosure, IL-2 and other leader sequences can be provided that are secreted from genetically modified T cells.

[0122] According to the present disclosure, treatment of cancers and tumors that express target antigens can use T cell therapeutics that express and secrete STARs, which can be combined with other immunotherapeutic agents. The present disclosure can be recombinant proteins that are delivered directly and can be combined with other immunotherapeutic agents.

[0123] According to the present disclosure, the article of manufacture can include one or more autologous or allogeneic T cells, STARs, and / or other immunotherapeutic agents expressed from or co-administered with the genetically modified gamma delta T cells.

[0124] Disclosed herein are gamma delta T cells, particularly genetically engineered T cells that secrete STAR agents.

[0125] Disclosed herein are gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins.

[0126] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins intended for secretion by including a modified serum albumin (mSA) signal peptide or a stem cell factor signal peptide to enhance production of the fusion proteins.

[0127] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins with optimized expression cassettes.

[0128] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins whose expression cassettes are optimized for expression in gamma delta T cells (γδ T cells).

[0129] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins whose expression cassettes are optimized for expression in gamma delta T cells harboring gamma 9 and delta 2 T cell receptor subunits.

[0130] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete a STAR agent, wherein the therapeutic agent is a bispecific T cell actuator.

[0131] Disclosed herein are gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins, wherein the synthetic fusion proteins are bispecific T cell actuators.

[0132] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins, wherein the synthetic fusion proteins are bispecific T cell actuators that include an anti-CD3 scFv fused to an scFv capable of binding to at least one of CD19, PTK7, GD2, SSTR2, and / or alpha-V beta-3 integrin.

[0133] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators encapsulating an anti-CD3 scFv fused to a cognate receptor ligand domain.

[0134] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete synthetic fusion proteins, which are bispecific T cell actuators comprising an anti-CD3 scFv fused to a receptor ligand domain from stem cell factor (SCF), thrombopoietin (TPO), SSTR2, or SSTR5.

[0135] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete a synthetic fusion protein, a bispecific T cell actuator comprising an anti-CD3 scFv fused to two or more copies of the receptor ligands SSTR2 and / or SSTR5.

[0136] Disclosed herein are genetically engineered gamma delta T cells (γδ T cells) that secrete a synthetic fusion protein that is a bispecific T cell actuator comprising an anti-gamma delta TCR scFv fused to an scFv that binds at least one of CD19, PTK7, GD2, SSTR2, and / or alpha-V beta-3 integrin.

[0137] Disclosed herein are genetically engineered gamma delta T cells that secrete a synthetic fusion protein, a bispecific T cell actuator comprising an anti-gamma delta TCR scFv fused to a cognate receptor ligand domain.

[0138] Disclosed herein are genetically engineered gamma delta T cells that secrete synthetic fusion proteins that are bispecific T cell actuator anti-gamma delta T cells fused with receptor ligand domains from stem cell factor (SCF), thrombopoietin (TPO), SSTR2, or SSTR5.

[0139] Disclosed herein are genetically engineered gamma delta T cells that secrete synthetic fusion proteins that are bispecific T cell actuators comprising an anti-JAML scFv fused to an scFv that binds CD19, PTK7, GD2, SSTR2, or alpha-V beta-3 integrin.

[0140] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include an anti-JAML scFv fused to a cognate receptor ligand domain.

[0141] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include an anti-JAML scFv fused to a receptor ligand domain from stem cell factor (SCF), thrombopoietin (TPO), SSTR2, or SSTR5.

[0142] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include an anti-JAML scFv fused to two or more copies of the receptor ligands SSTR2 or SSTR5.

[0143] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include a cognate JAML ligand fused to an scFv that binds CD19, PTK7, GD2, SSTR2, or alpha-Vbeta-3 integrin.

[0144] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include a cognate JAML ligand fused to a cognate receptor ligand domain.

[0145] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include a cognate JAML ligand fused with a receptor ligand domain from stem cell factor (SCF), thrombopoietin (TPO), SSTR2, or SSTR5.

[0146] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, where the synthetic fusion proteins are bispecific T cell actuators that include a cognate JAML ligand fused to two or more copies of a receptor ligand for SSTR2 or SSTR5.

[0147] Disclosed herein are genetically engineered T cells that secrete a STAR agent, where the therapeutic agent is a cytokine.

[0148] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, wherein the synthetic fusion proteins include dual cytokines.

[0149] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, wherein the synthetic fusion proteins comprise dual cytokines, wherein the dual cytokines are one or more of IL2 and / or IL15 (e.g., IL2-IL2, IL2-IL15, IL15-IL15).

[0150] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, wherein the genetically engineered T cells are gamma delta T cells.

[0151] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, wherein the genetically engineered T cells are gamma delta T cells, and the gamma delta T cells have gamma 9 and delta 2 T cell receptor subunits.

[0152] Disclosed herein are genetically engineered T cells that secrete one or more synthetic fusion proteins, wherein the genetically engineered T cells are gamma delta T cells harboring gamma 9 and delta 2 T cell receptor subunits, produced by ex vivo expansion of peripheral blood mononuclear cells from healthy donors using a two-step culture method.

[0153] Disclosed herein are engineered T cells that secrete one or more synthetic fusion proteins, wherein the engineered T cells can be gamma delta T cells, and further wherein the gamma delta T cells can have gamma 9 and delta 2 T cell receptor subunits, and have been modified for synthetic protein production by lentiviral or retroviral vector transduction or mRNA transfection.

[0154] Disclosed herein are genetically engineered hematopoietic cells that secrete one or more synthetic fusion proteins for which expression cassettes have been optimized.

[0155] Disclosed herein is the optimization of expression and secretion of bispecific scFv-based antibodies or soluble ligands from genetically engineered gdT cells for autocrine / paracrine binding between gdT cells and target-expressing tumor cells.

[0156] Disclosed herein is the local expression and secretion of bispecific scFv-based antibodies or soluble ligands from genetically engineered γδ T cells.

[0157] Disclosed herein is the endogenous expression of bispecific scFv-based antibodies or soluble ligands from γδ T cells and adoptive cell transfer of γδ T cells to enhance T cell cytotoxic function.

[0158] Disclosed herein are unique features, including sequences optimized for γδ T cell expression and an IL-2 leader sequence for secretion from genetically engineered γδ T cells, as shown in the accompanying figures.

[0159] Disclosed herein is the treatment of target-positive tumors with off-the-shelf γδ T cell therapeutics expressing bispecific scFv-based antibodies or soluble ligands.

[0160] Disclosed herein are recombinant vectors that encode one or more of the synthetic fusion proteins disclosed herein.

[0161] The present specification discloses a recombinant vector, which can be any vector known to those skilled in the art, including but not limited to a recombinant lentiviral vector or a recombinant retroviral vector, encoding one or more synthetic fusion proteins disclosed herein. The recombinant vector (e.g., lentiviral or retroviral) can include an internal promoter. The internal promoter can be, for example, an MND or HSPA8 promoter.

[0162] Disclosed herein are recombinant lentiviral or retroviral vectors encoding one or more fusion proteins disclosed herein and further encoding short hairpin RNAs targeting beta2 microglobulin (B2M) (SEQ ID NO: 150) and / or class II transcription activator (CIITA) (SEQ ID NO: 151). The vectors may optionally include the internal promoter MND or HSPA8.

[0163] Disclosed herein are methods for preparing engineered T cells that secrete one or more synthetic fusion proteins, comprising: (a) expanding healthy donor immune cells (e.g., g9d2 T cells) (see FIG. 6).

[0164] The disclosed genetically engineered cells can be applied as cancer therapeutics that can treat cancers including, but not limited to, B-cell malignancies, neuroblastoma, osteosarcoma, neuroendocrine tumors (NETs), and acute myeloid leukemia (AML).

[0165] Disclosed herein are expression vectors for engineering cells to express the therapeutic agents or synthetic fusion proteins disclosed herein.

[0166] Disclosed herein is a method for treating cancer, comprising: (a) culturing and expanding PBMCs from a third-party, healthy donor ex vivo in the presence of IL-2 and zoledronic acid to enrich for gdT cells; and (b) genetically modifying the enriched gdT cells ex vivo for STAR expression and secretion. The genetically modified gdT cells are qualified and can be frozen and banked for subsequent use or immediately infused into a recipient to control and / or reduce tumor growth.

[0167] Disclosed herein is a therapeutic agent comprising a single chain antibody variable domain that binds to SSTR2, wherein the single chain antibody variable region that binds to SSTR2 has an amino acid sequence that is at least 96% identical to SEQ ID NO:156.

[0168] Disclosed herein is a therapeutic agent comprising a single chain antibody variable domain that binds to GD2-3, wherein the single chain antibody variable region that binds to GD2-3 has an amino acid sequence that is at least 96% identical to SEQ ID NO:161.

[0169] Disclosed herein is a therapeutic agent having, from N- to C-terminus, a gamma delta T cell optimized signal peptide that is cleaved prior to secretion, a linker that is SEQ ID NO: 155 (G4S), and a T cell binding protein that is SEQ ID NO: 94 (HUM2).

[0170] Disclosed herein are therapeutic agents wherein a single chain antibody variable domain that binds SSTR2 is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO:157, 158, 159, or 160.

[0171] Disclosed herein are therapeutic agents wherein a single chain antibody variable domain that binds GD2-3 is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO:162, 163, 164, or 165.

[0172] Disclosed herein are engineered gamma delta T cells capable of secreting at least one therapeutic protein, the therapeutic protein comprising, from N- to C-terminus, a gamma delta T cell-optimized signal peptide, a tumor cell binding domain, a linker, and a T cell binding domain, wherein the tumor cell binding domain has an amino acid sequence at least 96% identical to SEQ ID NO: 156 or SEQ ID NO: 161. The engineered gamma delta T cells optionally comprise a linker having the sequence of SEQ ID NO: 155 (G4S). The engineered gamma delta T cells optionally comprise a T cell binding protein having the sequence of SEQ ID NO: 94 (HUM2).

[0173] In one variation, the engineered gamma delta T cell has a tumor cell binding domain encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 157, 158, 159, or 160.

[0174] In one variation, the engineered gamma delta T cell has a tumor cell binding domain encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 162, 163, 164, or 165.

[0175] Disclosed herein is a recombinant viral vector encoding a therapeutic protein that can be biosynthesized and secreted by gamma delta T cells, the therapeutic protein comprising, from N- to C-terminus, a gamma delta T cell-optimized signal peptide that is cleaved prior to secretion, a tumor cell binding protein domain, a linker, and a T cell binding protein, wherein the tumor cell binding protein domain binds to either SSTR2 or GD2-3, and the tumor cell binding protein domain that binds SSTR2 has an amino acid sequence at least 96% identical to SEQ ID NO: 156, and the tumor cell binding protein domain that binds GD2-3 has an amino acid sequence at least 96% identical to SEQ ID NO: 161. In one variation, the linker is SEQ ID NO: 155. In a further variation, the T cell binding protein is SEQ ID NO: 94.

[0176] In one variation, the tumor cell binding protein domain that binds SSTR2 is encoded by a nucleic acid sequence at least 96% identical to at least one of SEQ ID NOs: 157, 158, 159, or 160.

[0177] In one variation, the tumor cell binding protein domain that binds GD2-3 is encoded by a nucleic acid sequence at least 96% identical to at least one of SEQ ID NOs:162, 163, 164, or 165.

[0178] Experimental data Provided herein are optimized STAR constructs capable of improving expression from gamma delta T cells. The unique ECOg-optimized molecules of the present disclosure are shown to provide improved expression in gamma delta T cells over standard human codon optimization. Figure 15. Expression improvements applied to gamma delta T cells are reflected in improved or enhanced cytotoxicity. ECOg provides improvements over standard human codon optimization in gamma delta-mediated gene expression. Here, gdT cells were transfected with mRNA carrying ECOg- or human (HCO)-optimized hSCF-encoding STARs. Four hours after transfection, gdT cells (effector) were co-incubated with CMK cells (target), and the cytotoxicity of the transfected gdT against CMK cells was measured.

[0179] This ECOg optimization is applied to optimize elements of STARs associated with multiple steps along the expression pathway, providing highly efficient therapeutic effects through improved cytotoxicity over non-optimized cells. Figure 1 provides a schematic diagram of an exemplary STAR framework. By construct, STARs disclosed through the sequence listing provided below are optimized to improve expression over corresponding human codon-optimized and / or non-optimized constructs. The provided STAR constructs are specifically optimized for improved expression in gamma delta T cells. This optimization improves the ability of gamma delta T cells to more efficiently produce and express therapeutic proteins, as demonstrated by enhanced cytotoxic activity.

[0180] This optimization targets each step in the protein production pathway, aiming to improve translation and expression in the gamma delta T cell environment. For example, promoter elements of STAR have been optimized to improve gamma delta T cell expression of therapeutic molecules. For example, the ECO-optimized MND promoter (SEQ ID NO: 152) is provided herein. In one variation, the ECO-optimized non-viral promoter, the HSP8 promoter (SEQ ID NO: 153), is provided herein. See Figures 12 and 13 for data demonstrating sustained GFP expression in cells driven by the disclosed novel HSP8 promoter compared to the MND promoter. MND and HSPA8 GFP expression in lentivirally transduced gdT cells. Fresh gdT cells were transduced with lentiviral particles carrying a GFP expression cassette driven by either the MND (myeloproliferative sarcoma virus MPSV enhancer, negative control region NCR deletion) or HSPA8 (heat shock 70 kDa protein 8). GFP+ cells were measured on day 6 of gdT cell expansion (Figure 12). The mean fluorescence intensity (MFI) of all cells was measured on day 6 of gdT cell expansion (FIG. 13).

[0181] Disclosed herein is a novel HSPA8 promoter (SEQ ID NO: 153) that has been optimized to provide a non-viral promoter that can achieve expression levels comparable to the viral promoter MND. Together with the HSPA8 promoter, disclosed herein is a STAR with a preferred composition that uses a non-viral promoter to reduce known problems caused by the MND viral promoter. As shown in Figures 12 and 13, the novel HSPA8 promoter has been optimized to achieve expression levels comparable to the MND promoter, providing a desired non-viral promoter.

[0182] Through optimization of the signal peptide components, STARs have been tailored for expression in gamma delta T cells. The increased expression resulting from the optimized signal peptide components was demonstrated by improved cytotoxicity of STAR-expressing cells. In one variation, the mSA signal peptide (SEQ ID NO: 3) was optimized for gamma delta T cell expression. The disclosed signal peptide improved the cytotoxicity of gamma delta T cells through increased expression. Figure 14 shows that the mSA signal peptide improves the cytotoxicity of secreted media from 293T cells expressing PTK7 and GD2 STARs. Here, 293T cells were transfected with each STAR, and conditioned media was collected 48 hours post-transfection. Gamma delta cells (effector) and IMR5 cells (target) were co-incubated in the presence of conditioned media, and the percentage killing of IMR5 cells was measured after 4 hours. The ratio of effector cells to target cells is indicated on the X-axis label.

[0183] In one variation of STAR, expression-optimized linkers (e.g., flexible linkers or central linkers) are provided that are specifically optimized for improved secretion from gamma delta T cells. Two optimized linkers are provided herein: albumin linkers (SEQ ID NOS: 31-37) and Fc linkers (SEQ ID NOS: 87-93).

[0184] Figures 16 and 17 demonstrate the improvements provided by the disclosed linkers. Here, albumin fusions and mSA signal peptides increase STAR secretion over IL2 designs. 293T cells were transfected with various STAR designs, and conditioned media was collected 48 hours post-transfection. Figure 16 provides Western blot analysis of the designed STAR proteins. Figure 17 shows quantification of STAR secretion in the media normalized to the IL2 PTK7 CD3 STAR design.

[0185] Figure 18 further characterizes the improvement provided by the disclosed optimized albumin linkers, also referred to as albumin fusions. Figure 18 shows that central albumin fusions improve STAR secretion. To address the potential steric hindrance of N-terminal albumin fusions on STAR proteins, albumin fusions were tested placed between the tumor-targeting and gdT-targeting ends of the STAR. 293T cells were transfected with IL2 SSTR2 LH STAR, which has a centrally positioned albumin molecule, and with IL2 SSTR2 LH STAR, which does not have a centrally positioned albumin molecule. Conditioned media was collected 48 hours post-transfection and subjected to quantitative Western blot analysis.

[0186] The following data demonstrate the cytotoxic activity of the disclosed STAR variants.

[0187] Figure 19. gdT cells transduced with STAR-encoding lentivirus acquire cytotoxicity against target cells. Here, fresh gdT cells were transduced with lentiviral particles carrying IL2CD19LHCD3STAR (e.g., SEQ ID NOs: 1-2). After several days of cell growth, transduced and mock-transduced gdT cells (effector [E]) were co-incubated with 697 cells (target [T]), and toxicity against 697 cells was measured. The ratio of E cells to T cells is shown on the X-axis. This demonstrates that gdT cells transduced with STAR-encoding lentivirus acquire cytotoxicity against target cells.

[0188] Figure 20. Integrin aV B3 CD3 STAR (e.g., SEQ ID NOs: 45-51) promotes target cell killing. (A) 293T cells were transfected with a plasmid expressing Integrin aV B3 CD3 STAR, and conditioned medium supernatants were collected 48 hours post-transfection. Western blots of conditioned medium showed the correct size of Integrin aV B3 STAR. (B) Fresh gdT cells were transfected with RNA encoding Integrin aV B3 CD3 STAR. gdT cells (effector [E]) were mixed with human erythroleukemia (HEL) cells (target [T]), and cytotoxicity against HEL cells was measured. The ratio of effector to target cells is shown along the X-axis.

[0189] Figure 21. IL2CD19CD3 STAR (e.g., SEQ ID NOS: 1-2) promotes target cell killing. Here, 293T cells were transduced with lentivirus encoding GFP or IL2CD19CD3 STAR (e.g., SEQ ID NOS: 1-2) driven by either the HSPA8 or MND promoter. Conditioned medium was collected 48 hours post-transduction. gdT cells (effector [E]) and 697 cells (target [T]) were mixed, conditioned medium was added, and cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the x-axis. This figure demonstrates the efficacy of both IL2CD19CD3 STAR (e.g., SEQ ID NOS: 1-2) variants, demonstrating that the novel HSPA8 promoter offers a non-viral alternative comparable to the MND promoter.

[0190] Figures 22 and 23 show that mSA PTK7 CD3 STAR (e.g., SEQ ID NO: 3, SEQ ID NOs: 17-23) promotes target cell killing. Here, 293T cells were transfected with a plasmid expressing mSA PTK7 CD3 STAR (Figure 22). Conditioned medium was collected 48 hours post-transfection. Western blot of the conditioned medium showed a product of the expected size. In Figure 23, gdT cells (effector [E]) and IMR5 cells (target [T]) were mixed, conditioned medium was added, and cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the x-axis.

[0191] Figures 24 and 25 show that mSA (SEQ ID NO: 3) and the native signal peptide hSCF CD3 STARs promote target cell killing. Figure 24 shows that 293T cells were transfected with a plasmid expressing SCF CD3 STAR using the native SCF signal peptide (hSCF STAR). Conditioned medium was collected 48 hours post-transfection. Western blot of the conditioned medium showed a product of the expected size. Figure 25 shows that gdT cells (effector [E]) were transfected with mRNA encoding mSA (SEQ ID NO: 3) and the native signal peptide version of hSCF CD3 STAR and mixed with IMR5 cells (target [T]). Cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the X-axis.

[0192] Figures 26 and 27 show that mSA and IL2 GD2 (e.g., SEQ ID NOS: 38-44) CD3 STARs promote target cell killing. In Figure 26, 293T cells were transfected with plasmids expressing mSA or IL2 GD2 HL CD3 STAR. Conditioned medium was collected 48 hours post-transfection. Western blot of the conditioned medium showed products of the expected size. In Figure 27, gdT cells (effector [E]) and IMR5 cells (target [T]) were mixed, conditioned medium was added, and cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the x-axis.

[0193] Figures 28 and 29 show that IL2 SSTR HL and LH CD3 STAR promote target cell killing. In Figure 28, 293T cells were transfected with plasmids expressing the heavy / light (HL) and light / heavy (LH) configurations of the SSTR2 scFv in the IL2 SSTR2 CD3 STAR chassis. Conditioned medium was collected 48 hours post-transfection, and Western blots detected bands of the appropriate size. In Figure 29, gdT cells were transfected with mRNA encoding IL2 SSTR2 HL or LH CD3 STAR. Transfected gdT cells (effector [E]) were mixed with IMR5 cells (target [T]), and the resulting cytotoxicity against the target cells was measured.

[0194] Figures 30 and 31 show that humanized / deimmunized versions of CD3 scFv direct gdT-mediated killing. In Figure 30, 293T cells were transfected with plasmids expressing CD3 (control) and Hum2 scFv (e.g., SEQ ID NOS: 94-100) (humanized / deimmunized) versions of IL2 SSTR2 LH STAR. Conditioned medium was collected 48 hours post-transfection. Western blot of the conditioned medium showed a product of the expected size. In Figure 31, gdT cells (effector [E]) were co-incubated with IMR5 cells (target [T]) in the presence of conditioned medium. Cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the X-axis.

[0195] Figures 32 and 33 show lentiviral delivery of shRNAs that knock down surface expression of HLA class I and II. In Figure 32, gdT cells were transduced with concentrated and non-concentrated LentET vectors carrying a cassette driving expression of an anti-B2m shRNA (B2m shRNA1), and the percentage of B2m (HLA class I) knockdown was measured by flow cytometry. In Figure 33, gdT cells were transduced with a LentET vector carrying a cassette driving expression of an anti-CIITA shRNA (CIITA shRNA7), and the percentage of CIITA (HLA class II) knockdown was measured by flow cytometry.

[0196] The STARs disclosed herein are uniquely engineered to preemptively address and avoid HLA mismatches to improve graft survival. In one variation, the gdT cell products (STARs) disclosed herein are allogeneic, meaning they are derived from non-donor PBMCs. Generally, graft-versus-host disease (GVHD) is not thought to be a major issue due to the MHC-independent killing of gdT cells. However, given that these gdT cell therapies will likely be administered to patients who are already severely immunosuppressed, erring on the side of caution, we take proactive steps herein to ensure immunogenicity is not an issue and attempt to make the cell products more "universal."

[0197] Current literature suggests that immunogenicity can be reduced by reducing (siRNA / shRNA) or eliminating (CRISPR knockout) the presence of HLA I and II complexes on the donor cell surface. The presence of multiple HLA genes, most of which are highly polymorphic, presents unique challenges. However, non-polymorphic protein targets also exist, the suppression of which results in the elimination or reduction of HLA expression on the cell surface. For HLA I, we use lentiviral vectors expressing shRNAs targeting the non-polymorphic β chain of all HLA class I surface complexes, also referred to as β2-microglobulin. By reducing the levels of B2M, an essential component of the HLA I complex, we also reduce HLA I expression on the cell surface. Reducing HLA class II expression is not as straightforward. While all HLA II complexes share a common structural element that can be targeted for knockdown, literature suggests that targeting the "class II major histocompatibility complex transcriptional activator" (CIITA) can universally reduce or eliminate HLA II expression on the cell surface. CIITA is required for transcription of the HLA II gene; without it, transcription does not occur. Similar to HLA I, we use a lentiviral vector encoding an shRNA against CIITA to reduce the surface expression of HLA II. Finally, a lentiviral vector producing STAR, as well as shRNAs against both B2M and CIITA, could reduce the levels of HLA I and II on the surface of gdT cells, resulting in a safer and more effective treatment, siLentET.

[0198] Figures 34 and 35 show that alternative gdT-targeting moieties direct gdT-mediated cytotoxicity. In Figure 34, 293T cells were transfected with plasmids expressing STAR directed to target cells with anti-GD2 scFv and anti-gdT TCR scFvs (gd-c V1 and gc-c V6) or anti-CD3 scFv directed to gdT cells. Conditioned medium was collected 48 hours post-transfection. In Figure 35, gdT cells (effector [E]) were co-incubated with IMR5 cells (target [T]) in the presence of conditioned medium. Cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the X-axis.

[0199] Figures 36 and 37 show somatostatin ligand gdT-mediated cytotoxicity against NET cells. In Figure 36, 293T cells were transfected with a plasmid expressing STARs that target target cells with the somatostatin ligand (SST28) and gdT cells with an anti-CD3 scFv. Conditioned medium was collected 48 hours after transfection. In Figure 37, gdT cells (effector [E]) were co-incubated with IMR5 cells (target [T]) in the presence of conditioned medium. Cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the X-axis.

[0200] Figure 38 shows IL2 TPO BR CD3 STAR expression, in which a plasmid carrying IL2 TPO BR CD3 STAR was transfected into 293T cells and expression of the expected protein product was detected by Western blot.

[0201] Figure 39 shows that mRNA-mediated protein expression correlates with mRNA free energy. Here, Jurkat cells were transfected with a series of optimized mRNA constructs of ECOg encoding a range of low to high mRNA free energies. GFP expression was measured 8 hours after transfection.

[0202] In Figure 40, 293T-17 cells were transfected with PTK7-14 and SSTR2-8 STAR plasmids, and conditioned media was collected 48 hours post-transfection. IMR5 tumor target model cells were co-incubated with gamma delta T cells at a 1:1 or 5:1 ratio in the presence of STAR-containing conditioned media. Percent target cell killing was determined by flow cytometry. 7AAD refers to 7-aminoactinomycin D, a dye used to assess cell death. Annexin is used to measure apoptosis or cell death.

[0203] In Figure 41, 293T-17 cells were transfected with STAR-expressing plasmid DNA. Conditioned medium was collected 48 hours post-transfection and analyzed for the presence of STAR proteins by anti-His Western blot assay. Column 1: Blank. Column 2: mSA PTK7-4 CD3-2 His. Column 3: mSA PTK7-13 CD302 His. Column 4: Blank. Column 5: IL2 SSTR2-3 CD3-2 His. Column 6: IL2 SSTR2-8 CD3-2 His. Column 7: mSA PTK7-4 CD3-2 His. Column 8: mSA PTK7-14 CD3-2 His. Column 9: Blank.

[0204] In Figure 42, 293T-17 cells were transfected with the gd2-3 plasmid and conditioned medium was collected 48 hours post-transfection. IMR5 tumor target model cells were co-incubated with gamma delta T cells at a 1:1 or 5:1 ratio in the presence of STAR-containing conditioned medium. Percent target cell killing was determined by flow cytometry.

[0205] In Figure 43, SSTR-STAR conditioned medium was prepared by transient transfection in 293T cells and quantified by ELISA. Gamma delta T cells and IMR5 cells were co-incubated in the presence of various concentrations of STAR protein. Cytotoxicity was determined by flow cytometry.

[0206] In Figure 44, SSTR-STAR conditioned medium was prepared by transient transfection in 293T cells and quantified by ELISA. Gamma delta T cells and IMR5 cells were co-incubated in the presence of various concentrations of STAR protein. Cytotoxicity was determined by flow cytometry.

[0207] Figure 45 shows the cytotoxicity of various PTK7-STAR proteins (x-axis) at a 1:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. STAR-conditioned medium was prepared by transient transfection in 293T cells. Gamma delta T cells and IMR5 cells were co-incubated in the presence of STAR proteins at various effector (gamma delta T cells) to target (IMR5 cells) ratios. Cytotoxicity was determined by flow cytometry.

[0208] Figure 46 shows the cytotoxicity of various PTK7-STAR proteins (x-axis) at a 5:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. STAR-conditioned medium was prepared by transient transfection in 293T cells. Gamma delta T cells and IMR5 cells were co-incubated in the presence of STAR proteins at various effector (gamma delta T cells) to target (IMR5 cells) ratios.

[0209] Figure 47 shows the cytotoxicity of various SSTR-STAR proteins (x-axis) at a 1:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. SSTR-STAR conditioned medium was prepared by transient transfection in 293T cells. Gamma delta T cells and IMR5 cells were co-incubated in the presence of STAR proteins at various effector (gamma delta T cells) to target (IMR5 cells) ratios. Cytotoxicity was determined by flow cytometry.

[0210] Figure 48 shows the cytotoxicity of various SSTR-STAR proteins (x-axis) at a 5:1 effector (gamma delta T cells) to target (IMR5 cells) ratio. SSTR-STAR conditioned medium was prepared by transient transfection in 293T cells. Gamma delta T cells and IMR5 cells were co-incubated in the presence of STAR proteins at various effector (gamma delta T cells) to target (IMR5 cells) ratios. Cytotoxicity was determined by flow cytometry.

[0211] Figure 49 shows the cytotoxicity of additional SSTR-STAR proteins (x-axis) at effector (gamma delta T cells) to target (IMR5 cells) ratios of 1:1 and 5:1. SSTR2 cytotoxicity assay - 293T cells were transfected with a STAR-encoding plasmid. Conditioned medium was collected 48 hours post-transfection. Gamma delta T cells (effector) were co-incubated with IMR5 cells (target) at effector:target ratios of 1:1 and 1:5. Cytotoxicity was determined by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0212] method This disclosure provides methods and compositions for genetically engineered gamma delta T cells that express and secrete STARs for the treatment of various cancers and solid tumors. See Figure 9. Gamma delta T cells can be expanded from autologous or allogeneic donors under serum-free conditions. Donors can be selected based on a range of screening criteria, including, but not limited to, disease-specific / target-specific profiles, such as cytotoxicity assays in the presence or absence of other drugs and / or immunotherapies (Figure 8). Gamma delta T cell expansion can be performed under serum-free conditions in a two-phase expansion procedure consisting of T-flask-based culture in IL-2 and zoledronic acid from days 0 to 6 (Phase 1), and Bioreactor-based culture in IL-2 from days 6 to 12 (Phase 2). An αβ T cell depletion step can be performed on day 6 of culture prior to Bioreactor-based culture. Viral-based genetic modification (lentivirus and / or gamma retrovirus) can be performed in either phase 1 or phase 2 of gamma delta T cell expansion. Alternatively, AAV- or mRNA-based genetic modification can be performed in the second phase of gamma delta T cell expansion for the expression and secretion of other immunomodulatory substances, such as STARs and / or IL2-IL5 bispecific molecules.

[0213] In the present disclosure, the production of genetically engineered gamma delta T cells from autologous or allogeneic donor PBMCs under serum-free conditions can be performed in a two-phase expansion procedure: genetic modification can be performed during either the first or second phase of gamma delta T cell expansion.

[0214] Donor pre-screening can be performed based on a set of criteria that allow for optimal growth, genetic modification, and cytotoxicity. Donor pre-screening can be based on disease-specific selection criteria, such as disease / target-specific cytotoxicity assays in the presence or absence of other drugs and / or immunotherapies.

[0215] According to the present disclosure, the present manufacturing method can solve the problems of serum-free growth conditions, donor screening for optimal growth, genetic modification, and cytotoxicity against disease-specific targets. According to the present disclosure, the two-phase expansion procedure can include a step of depleting αβ T cells for an optimal product safety profile. Genetic modification can be performed in either the first or second phase, or a combination thereof, for optimal secretion of STARs and / or other immunomodulators, such as IL2-IL5 bispecific molecules.

[0216] In aspects of the present disclosure, the expansion and genetic modification of gamma delta T cells allows for the expression and secretion of STARs and / or other immunomodulatory substances with enhanced disease-specific efficacy.

[0217] According to the present disclosure, the methods can include: serum-free expansion conditions in a two-phase expansion method, genetic modification in either phase of expansion for combinatorial engineering with STARs and / or immunomodulators, expression and secretion of immunomodulators that promote the proliferation and viability of gamma delta T cells both in vitro and in vivo.

[0218] For the treatment of cancer and solid tumors, autologous or allogeneic gamma delta T cells engineered with STARs harboring target tumor antigens may be provided by the present disclosure.

[0219] According to the present disclosure, the following may be provided: biphasic growth; genetic modification in either or both phases of growth; serum-free growth conditions; and endogenously expressed and secreted IL2-IL5 bispecific molecules to enhance growth, viability, and function.

[0220] In FIG. 8, a method according to the present disclosure is disclosed and summarized below. Process 1 Isolating PBMCs from leukopak (day 0) Process 2: Seeding PBMCs into T-flasks (Day 0) Process 3 : Change the medium (day 3) Process 4 : Depletion of αβ T cells (day 6) Process 5 : Seeding gdT cells into the bioreactor (Day 6) Process 6 : IL-2 addition (day 9) Process 7 : Cell collection and cryopreservation (day 12) Complete leukopaks manufactured by the American Red Cross typically contain 1-1.5e10 total nucleated cells per leukopak and have a total volume of 150-400mL.

[0221] procedure: Day 0 Process 1PBMC isolation from leukopaks (Day 0). This step involves the following: draw 22 mL of whole blood and add 18 mL of Ficoll-Paque to 50 mL conical tubes (two per donor). Remove blood from the collection tubes and pipette it into the 50 mL conical tubes for each donor. Record the starting blood volume. This volume does not include the PBS wash volume in the next step. Rinse the collection tubes with 4 mL of PBS to remove any blood and cells remaining on the sides of the tube, then place them into the 50 mL conical tube with the remaining blood. Add more PBS to achieve a final PBS:blood volume ratio of 1:1. Total blood volume / donor after dilution = 44 mL. Gently mix the blood by inverting the 50 mL conical tube. Carefully add 22 mL of diluted blood on top of the 18 mL of Ficoll-Paque medium (do not mix). Spin the conical tube at 400 x g for 35 minutes at 20°C. Remove the top layer (plasma) with a pipette. Collect the PBMC layer with a pipette and transfer to a new 50 mL conical tube containing pooled samples from the same donor. Wash the cells by suspending them in 3 times the volume of the pellet (approximately 45 mL) of PBS, then spin at 500 x g for 10 minutes at 20°C. Resuspend in 10 mL of PBS and wash again. Spin at 200 x g for 5 minutes at 20°C. Resuspend in 10 mL of PBS and wash again. Spin at 200 x g for 5 minutes at 20°C. Resuspend in 10 mL of PBS and wash again. Spin at 200 x g for 5 minutes at 20°C.

[0222] Process 2 : Seed PBMCs in T-flasks (day 0). This step involves the following steps: Resuspend cell pellet in 10 mL of OpTmizer medium. Count cells. Set aside 1 mL (500K) of cells for immunotyping CD3 / gdTCR. Include gdTCR FMO control from pooled samples. Use approximately 100K cells / tube. Measure cell density in the OpTmizer to a final cell density of 1.5x10 6Culture the cells at 37°C / 5% CO2. Add IL-2 and zoledronic acid to achieve a concentration of 500 IU / mL IL-2 and 5 μmol / L zoledronic acid. Tilt the T-75 flask (standing upright) so that the cell culture medium is concentrated in the lower half of the flask. Culture the cells at 37°C / 5% CO2.

[0223] Day 3. Process 3 : Medium change (Day 3). This step involves the following steps: Transfer cells to a 50 mL conical tube and gently pipette up and down with a 10 mL pipette to break up clumps of cells. Centrifuge cells at 250 x g for 10 minutes at room temperature. Resuspend cells in OpTmizer medium. Count cells. Count cells in complete OpTmizer supplemented with 500 IU / mL IL-2 and 5 μmol / L zoledronic acid until a final cell density of 1.5 x 10 6 cells / mL same Re-plating into a T-75 flask. To concentrate the cell culture medium in the lower half of the T-75 flask, culture the cells in a tilted flask (standing upright) at 37°C / 5% CO2. Lentiviral / retroviral transduction. After step 4, transduce the cells to a final cell density of 1.5 x 10 in a complete OpTmizer supplemented with lentivirus / retrovirus at the desired TU / mL in the presence of 500 IU / mL IL-2 and 5 μmol / L zoledronic acid, plus transduction enhancer. 6 cells / mL same The cells are replated into a T-75 flask. The flask is tilted (standing upright) so that the cell culture medium is concentrated in the lower half of the T-75 flask, and the cells are cultured at 37°C / 5% CO2.

[0224] Day 4. This process involves the following steps: Perform a second transduction at the same TU / mL by removing half of the cells and transferring them to a 50 mL conical tube. Centrifuge the cells at 250 x g for 10 minutes at room temperature. Transduce the cells to a final cell density of 1.5 x 10 in a complete OpTmizer supplemented with lentivirus / retrovirus at the desired TU / mL in the presence of 500 IU / mL IL-2 and 5 μmol / L zoledronic acid and transduction enhancer. 6 cells / mL same The cells are replated into a T-75 flask. The flask is tilted (standing upright) so that the cell culture medium is concentrated in the lower half of the T-75 flask, and the cells are cultured at 37°C / 5% CO2.

[0225] Day 5. This process involves the following steps: Transfer cells to a 50 mL conical tube; Centrifuge cells at 250 x g for 10 minutes at room temperature; Resuspend cells in a complete OpTmizer supplemented with 500 IU / mL IL-2 and 5 μmol / L zoledronic acid; same The cells are replated into a T-75 flask. The flask is tilted (standing upright) so that the cell culture medium is concentrated in the lower half of the T-75 flask, and the cells are cultured at 37°C / 5% CO2.

[0226] Day 6. Process 4 : Depletion of αβ T cells (day 6). This step involves the following steps: Transfer cells to a sterile container. Count the cells. Remove 5e6 cells and save for flow immunotyping. Perform αβ T cell depletion with CliniMACS Plus. Process 5 : Seed gdT cells into Bioreactor 500M-CS (day 6). This process involves the following steps: Resuspend cells in OpTmizer medium. Count cells. Add 2e9 viable cells to each Bioreactor 500M-CS containing 2.5L of complete OpTmizer medium supplemented with 1000IU / mL IL-2. Culture cells at 37°C / 5% CO2.

[0227] Day 9. Process 6: IL-2 Addition (Day 9). This step involves the following steps: Add 1000 U / mL of IL-2 through the needle port / tubing at the top of each bioreactor device. Return the GRex devices to the incubator at 37°C / 5% CO2.

[0228] Day 11. AAV transduction. This step involves the following: Reduce the medium in the GRex device to 1 / 2 to 3 / 4 of the culture volume. Add the intended AAV VG / cells supplemented with the intended transduction-enhancing agent directly to the GRex. Return the GRex device to the incubator at 37°C / 5% CO2.

[0229] Day 12. Process 7 : Cell Harvesting and Cryopreservation (Day 12). This process involves the following steps: Using the Gather-Rex pump, remove the top 2 L (80%) of medium from each Bioreactor device without disturbing the cell layer on the Bioreactor membrane. Recover the cells in the remaining 500 mL of medium and transfer the cells to a 500 mL centrifuge bottle. Set aside a sample of cells for immunotyping analysis, count the number of live cells, and determine viability with trypan blue. Centrifuge the cells at 300 x g for 20 minutes at room temperature. Resuspend the cells in cryopreservation medium (PBS + 5% HSA + 10% DMSO) to 10e6 cells / mL. Distribute the cells into cryobags. Place the bags in a control rate freezer and run the specified freezing program. After the freezing program is complete, transfer the cryobags to liquid nitrogen for storage.

[0230] Electroporation of mRNA. This step involves the following: After step 7#4 (centrifugation), resuspend cells in electroporation buffer and add mRNA. Electroporate cells using appropriate instrument settings. Cells are returned to complete medium and either incubated for 2 hours at 37°C / 5% CO2 or immediately resuspended in cryo-medium for freezing, as described in step 7#5. [Example]

[0231] Example 1 Gamma delta T cells were thawed. Cells were incubated in complete medium at 37°C for 2 hours. 1e7 cells were electroporated with 15µg of mRNA. After 24 hours, assay tubes were loaded with gdT cells + 50% conditioned medium for cytotoxicity assay against 697 cells.

[0232] Figures 9-11 show the experimental results. Figure 9 provides an overview of the gdT cell expansion process. Figure 10 shows the identification of donors capable of adequate ex vivo expansion of gdT cells from peripheral blood mononuclear cells (PBMCs). Figure 11 shows the results of screening ex vivo expanded gdT cells to identify donors that produce gdT cells that exhibit high cytotoxicity against K562 human cancer cells.

[0233] The procedure includes: the purpose : Test the cytotoxicity of gdT cells transfected with CD19-CD3 STAR mRNA against 697 cells, Nalm6 cells, and 697-CD19KO cells.

[0234] Experimental Overview : gdT cells were thawed, transfected with CD19-CD3 STAR mRNA, and cytotoxicity was observed in 697 and Nalm6 cells approximately 24 hours later.

[0235] material : 12-day-old gdT cells harvested from two 20e6 vials of ARC2387 Grifols HSA (25%) Sterile PBS Nalm6 cells 697L cells CD19_CD3 STAR mRNA CTS OpTmizer T cell expansion kit,Life Technologies,Cat#A1048501 **Requires approximately 25 mL of complete gdT medium (OpTmizer + supplements + glutamine + 1000 IU / mL IL-2) Combine 40 mL of OpTmizer T cell basal growth medium with 1.106 mL of OpTmizer supplement and add 400 μL of 200 mM L-glutamine (Gibco, Cat. #16777-162). Prepare fresh and store at 4°C for no more than 1 week. IL-2 (1000U / μL), stored at -80℃ Reconstitute 250 μg of IL-2 (Peprotech, Cat. #AF-200-02) in 100 mM acetic acid and add 16.25 mL of sterile 1% BSA. Aliquot and store at -80°C for long-term storage (up to 1 year) or at -20°C for short-term storage during expansion. For 100 mM acetic acid, dissolve 14.4 μL of glacial acetic acid (Sigma, Cat. #A6283) in 2.486 mL of PBS (Gibco, Cat. #14190250). For 1% BSA, dissolve 200 mg of BSA (Sigma, Cat. #A9647) in PBS to a final volume of 20 mL. CD19 / CD3 bispecific antibody, BPS Biosciences, item 100441-1, 0.82 mg / mL; for this experiment, the bispecific antibody was obtained from a 10 μL aliquot frozen at −80° C. This stock was diluted 1:100 in OpTmizer medium, and 2.4 μL of bispecific antibody was added to each 200 μL reaction tube for a final concentration of 100 ng / mL. OpTiMem medium (Fisher cat#31-985-062) 4mm cuvette Violet Proliferation Dye(VPD450,BD Cat.562158)

[0236] procedure : Thaw 1 vial (25e6 cells) in 5 mL of 5% HSA / PBS: Prepare thawing medium: 2 mL of 25% HSA, 8 mL of PBS. Warm to 37°C. Thaw the vial by transferring it directly from a -80°C to 37°C water bath. Thaw until only a few ice cubes remain. Transfer the cells to a 15 mL conical tube. 1 mL of pre-warmed thawing medium is added slowly (drop-wise over 30 seconds) to the cells while stirring. Add an additional 3 mL of thawing medium Spin at 250 x g for 5 minutes at room temperature. Resuspend cells in 8 mL of OpTmizer + 15.1 μg / mL of IL2 in a T25 flask. Incubate in a TC incubator for 2 hours. Two hours after thawing, cells are harvested and dispersed into single cells by pipetting up and down. Count cells and determine viability

[0237] Electroporation : Pre-warm and equilibrate 3 mL of OpTmizer complete medium containing 1000 IU / mL of IL-2 into each well of a 6-well plate. The gdT cells counted in step 8 above are centrifuged at 250 xg for 5 minutes at room temperature. Resuspend cells in Optimem media to 10e6 cells / mL. Add 1 mL of cells (=10e6 cells) to each of two 1.5 mL epi tubes. Centrifuge the above epitube at 250 x g for 5 minutes at room temperature. Remove the supernatant completely. Resuspend the cells in 100 μL of Optimem. Pipette the cells up and down 3-5 times to disrupt the cell pellet.

[0238] For the CD19-CD3 STAR group, add 10 μL of mRNA (=15 μg). Mix and place the cell / mRNA mixture into a 4 mm cuvette (add the entire mixture, approximately 130 μL). Perform electroporation using the following settings: square wave 500V Pulse length 5ms 1 pulse 4mm cuvette Approximately 500 μL of warmed media (from the 6-well plate) is added to the cuvette and the media / cells are transferred back to the 6-well plate.

[0239] For the untransfected group, use 3 mL of medium from the 6-well plate, resuspend the cells in an epi tube, and transfer them to the 6-well plate. TC incubator overnight.

[0240] Cytotoxicity assay Staining of target cells (697 cells and Nalm6 cells) Place 5e6 target cells into a 15 mL conical tube and spin down. Wash the cells twice with PBS to remove serum proteins. For unstained control cells, set aside 1 / 5 of the cells. Prepare VPD450: add 1 µL of 1 mM VPD450 stock to 1 mL of PBS and mix. Resuspend cells in 500 μL of VPD450 / PBS. Unstained control cells are resuspended in PBS. Incubate in a 37°C water bath for 10 minutes. Add 9 mL of PBS and spin down the cells at 300 x g for 5 minutes. Resuspend in 10 mL of PBS and spin down the cells at 300 x g for 5 min. Cells are resuspended in 2 mL of complete Optimizer medium plus 1000 U / mL IL2. Viable cells are counted using trypan blue. Add OpTmizer+IL2 as needed to achieve a cell concentration of 1e6 cells / mL. Incubate target and effector cells in a total volume of 200 µL per flow tube based on the table below:

[0241] Preparation of gdT cells Collect the gdT cells and conditioned medium from the multiwell plate and place them in a 15 mL conical tube. Wash the flask with PBS and collect this in a separate 15 mL conical tube. Spin at 300 x g for 5 minutes at room temperature. Remove the conditioned medium from the cell pellet and transfer to a new conical tube. The conditioned medium is spun at 500 xg for 10 minutes to remove cell debris. Filter the conditioned medium through a 0.45 µm filter attached to a 5 mL syringe. The filtered conditioned medium is stored at -20°C. Resuspend and pool the cell pellets (from the cell pellet under conditioned medium and the PBS wash of the well). Resuspend in 300 μL of OpTmizer (a volume small enough to ensure >5e6 cells / mL). Pipette up and down with a P1000 pipette to loosen cell clumps. Take 10 μL of cells and dilute 1:10 by adding to 90 μL of PBS. This 1:10 dilution of cells is counted with trypan blue. Add the required amount of OpTmizer medium to each group to achieve a cell concentration of 5e6 cells / mL.

[0242] Flow cytometry analysis of cell killing Target and effector cells are incubated in an aluminum foil-covered flow tube at 37° C. for 4 hours. Prepare a compensation tube! See below. Wash the cells with 1 mL of FACS buffer. Wash the cells once with 500 µL of Annexin V binding buffer. Resuspend in 100 µL of 1x Annexin V binding buffer containing 2.5 µL of Annexin V-APC for 20 min at room temperature. Add 3 μL of 7-ADD per tube and incubate for at least 10 minutes. Wash by adding 1 mL of Annexin V binding buffer per reaction (rxn). Spin down at 300 x g for 5 minutes. Decant and resuspend in 200 μL of Annexin V binding buffer (not FACS buffer!). Gating on VPD450-positive cells, a flow run is performed to determine the percentage of apoptotic and dead target cells.

[0243] Compensation tube: Comp1: Unstained - target cells not stained with VPD450. Comp2: Target cells stained with VPD450 (stained and unstained target cells mixed at a 1:1 ratio). Comp3:7-ADD stained target cells (1:1 mix of live and dead cells). Comp4: target cells stained with Annexin V-APC (1:1 live vs dead cells).

[0244] Example 2, Lentiviral Transduction This embodiment supports FIGS. 12, 13, 15, 19, 25, and 26.

[0245] Day 1 - Transduction #1 (1) Thaw frozen gdTC cells at day 12 of culture, (2) Thaw lentivirus on ice, (3) Thaw LentiBoost and IL-2, (4) Warm Optimizer medium to 37°C, (5) Count gdTC cells, (6) Dispense 750,000 cells per 1.5 mL tube, (7) Spin down gdT cells at 300g for 5 minutes, (8) Carefully pipette the medium from the gdT cells, (9) Replace the medium with 500 μL of designed or complete Optimizer medium for mock transduction, (10) Add 10.5 μL of LentiBoost+IL-2 master mix, (11) Mix by pipetting up and down three times and dispense into labeled wells of a 48-well plate, (12) Add PBS to all surrounding wells to prevent the plate from drying out, and (13) Incubate overnight at 37°C.

[0246] Day 2 - Transduction #2 (1) Pipette all samples from the wells into 1.5 mL Eppendorf tubes. (2) Spin down at 300 g for 5 minutes at room temperature. (3) Carefully pipette the medium from the gdTC cells. (4) Replace with 500 μL of the designed LV. (5) Add 10.5 μL of LentiBoost+IL-2 master mix. (6) Mix by pipetting up and down three times and dispense into labeled wells of a 48-well plate. (7) Add PBS to all surrounding wells to prevent the plate from drying out. (8) Incubate overnight at 37°C.

[0247] Day 3 - Medium Change (1) Thaw IL-2. (2) Warm Optimizer medium in a 37°C water bath. (3) Gently pipette the cells into a labeled 1.5 mL tube. (4) Spin down at 300 g for 5 minutes at room temperature. (5) Prepare IL-2-supplemented complete Optimizer medium. (6) Carefully pipette the medium from the gdTC cells. (7) Replace with 500 μL of complete Optimizer. (8) Pipette up and down once and dispense into labeled wells of a 48-well plate. (9) Add PBS to all surrounding wells to prevent the plate from drying out. (10) Incubate at 37°C.

[0248] Day 6 - Analysis Cells are removed from the wells and analyzed in an appropriate assay. See Figures 12 and 13 for data showing the retention of GFP expression in cells driven by the novel HSP8 promoter of the present disclosure compared to the MND promoter. MND and HSPA8 GFP expression in lentivirally transduced gdT cells. Fresh gdT cells were transduced with lentiviral particles carrying a GFP expression cassette driven by either MND (myeloproliferative sarcoma virus MPSV enhancer, negative control region NCR deletion) or HSPA8 (heat shock 70 kDa protein 8). The percentage of GFP+ cells was measured on day 6 of gdT cell expansion (Figure 12). The mean fluorescence intensity (MFI) of all cells was measured on day 6 of gdT cell expansion (Figure 13). Figure 25 shows gdT cells (effector [E]) were transfected with mRNA encoding the mSA (SEQ ID NO: 3) and native signal peptide versions of hSCF CD3 STAR and mixed with IMR5 cells (target [T]). Cytotoxicity against target cells was measured. The ratio of effector cells to target cells is shown along the X-axis.

[0249] Example 3. Preparation of 293T conditioned medium This embodiment supports Fig. 14, Figs. 16 to 18, Figs. 20 to 24, Figs. 27 to 31, Figs. 34 to 37, and Figs. 40 to 42.

[0250] Day 1 - Plating of producer cells (1) Trypsinize 293T-17 cells. (2) Aspirate the medium from the flask. (3) Wash with 10 mL of PBS. (4) Add 10 mL of TrypLe and immediately aspirate. (5) Incubate the dried flask for approximately 10 minutes. (6) Resuspend the cells in 10 mL of DMEM / F12 + 10% FBS and transfer to a 50 mL conical tube. (7) Add an additional 10 mL of DMEM / F12 + 10% FBS to dilute the cells. (8) Dilute the cells to 1e6 cells / mL. (9) Plate 2 mL / well in a 6-well plate. (10) Incubate the cells overnight.

[0251] Day 2 - Transfection (per well) (1) Add 84.66 μL of Optimem to a 1.5 mL tube. (2) Add 2.5 μg of plasmid to the Optimem tube. (3) Add 2.5 μL of PEI to the Optimem / DNA mix and vortex immediately. (4) Vortex every 5 minutes for 15 minutes. (5) Add the entire volume (84.88 μL) to each well, then gently shake to mix. (6) Place in an incubator overnight.

[0252] Day 3 - Medium Change Replace the medium of the transfected cells with 1 mL of DMEM / F12 + 1% FBS. Aspirate the medium from the cells and add 1 mL of medium to each well.

[0253] Day 4 - Recovery The supernatant is collected, clarified by spinning at 3000 xg for 10 minutes, and transferred to a tube again.

[0254] Example 4. Adhesion cytotoxicity assay This embodiment supports FIGS. 14, 15, 23, 27, 29, 31, 34, 36, 37, 40, and 42.

[0255] Staining of target cells (CMK cells or IMR5 cells) Place 5e6 target cells into a 15 mL conical tube and spin down. Wash the cells twice with PBS to remove serum proteins. For unstained control cells, set aside 1 / 5 of the cells. Add 1 µL of 1 mM VPD450 stock to 1 mL of PBS and mix. Resuspend cells in 500 μL of VPD450 / PBS. Resuspend unstained control cells in PBS. Incubate in a 37°C water bath for 10 minutes. Add 9 mL of PBS and spin down the cells at 300 x g for 5 minutes. Resuspend the cells in 10 mL of PBS and spin down at 300 x g for 5 minutes. Resuspend cells in 2 mL of complete opTmizer + 1000 U / mL of IL2. Viable cells are counted using trypan blue. Add OpTmizer+IL2 as needed to achieve a cell concentration of 1e6 cells / mL. Target and effector cells are incubated in a total volume of 200 μL in the flow tube.

[0256] Preparation of gdT cells Collect the gdT cells and conditioned medium from the multiwell plate and place them in a 15 mL conical tube. Wash the flask with PBS and collect this in a separate 15 mL conical tube. Spin at 300 x g for 5 minutes at room temperature. Remove the conditioned medium from the cell pellet and transfer to a new conical tube. The conditioned medium is spun at 500 xg for 10 minutes to remove cell debris. The filtered conditioned medium is stored at -20°C. Resuspend the cells in 300 μL of OpTmizer and pool. Pipette up and down with a P1000 pipette to loosen cell clumps. Take 10 μL of cells and dilute 1:10 by adding to 90 μL of PBS. This 1:10 dilution of cells is counted with trypan blue. Add OpTmizer medium as needed to achieve a cell concentration of 5e6 cells / mL for each group of gdT cells.

[0257] Annexin V / 7AAD staining Target and effector cells are incubated in an aluminum foil-covered flow tube at 37° C. for 4 hours. Wash the cells with 1 mL of FACS buffer. Wash the cells once with 500 µL of Annexin V binding buffer. Resuspend in 100 µL of 1x Annexin V binding buffer containing 2.5 µL of Annexin V-APC and 3 µL of 7AAD for 15 min at room temperature. Wash by adding 1 mL of Annexin V binding buffer per reaction. Spin down at 300 x g for 5 minutes. Decant and resuspend in 200 μL of Annexin V binding buffer. Perform flow cytometry and determine the percentage of apoptotic and dead target cells by gating on VPD450 positive cells (VPD450 p).

[0258] Example 5, Western Blot This embodiment supports FIGS. 16 to 18, 20, 22, 24, 26, 28, 30, 35, 38, and 41.

[0259] SDS-PAGE Prepare 500 mL of 1x SDS running buffer. 50 mL of 10x Tris-Glycine running buffer 450mL of dH2O 2 0 Total 500mL Combination: 4x Sample Buffer (4x LDS Sample Buffer + β-mercaptoethanol (12 µL per 100 µL sample buffer)) +H20 +Protein Sample Heat in a heat block at 95°C for 5 minutes. Chill on ice for 1 minute. While the samples are heating, prepare the gel and electrophoresis equipment. Remove the gel from the package Rinse the gel with H20 Remove the tape from the bottom of the gel Assemble the electrophoresis apparatus and fill the inner tank Add 200mL of 1xRB + antioxidant to the inner tank. Add the remaining 300 mL of 1xRB to the outer tank. Carefully remove the comb from the gel Rinse wells with running buffer using a transfer pipette Load the samples onto the gel, including a lane loaded with 5 μL of protein standard. A current of about 80 to 125 V is applied to the gel to perform electrophoresis (as a result, the initial current is about 20 to 40 mA, and the current at the end of electrophoresis is about 10 mA).

[0260] Transfer of proteins to membranes Prepare 800 mL of transfer buffer (TB): 32 mL of 25x transfer buffer (Invitrogen) 160 mL of methanol Up to 800mL of H20 Pour approximately 600 mL of TB into a glass dish. Immerse a gel-sized piece of nitrocellulose membrane into the TB-containing dish and allow it to soak in TB for at least 5 minutes. Disassemble the electrophoresis tank. Rinse the gel cassette with water. Prepare the gel / membrane "sandwich" as shown in Figure 1. Use one or two blotting pads (as shown) on each side of the sandwich to assemble. Be careful not to trap air bubbles between any of the materials when assembling this sandwich. With the wells facing up, open the plastic gel cassette with a gel knife and remove the top plastic piece. The top of the well is cut off and a piece of filter paper moistened with TB is placed on top of the gel. Invert the gel and run a gel knife through the groove at the bottom of the gel to release the gel from the plastic. The feet of the gel are cut off and a pre-soaked membrane is placed on top of the gel. Place a piece of filter paper moistened with TB on top of the membrane. Place the pre-soaked blotting pad into the transfer module. Place the filter paper / gel / membrane / filter paper in the same orientation on top of the transfer pad and place another pre-soaked transfer pad on top of the filter paper. After the module is assembled, it is placed in the gel box and secured with clamps. Place the TB inside the module so that it is just enough to cover the top surface of the pad. Pour water into the outer chamber (inside the gel box). Set the voltage to 15V (constant) and transfer for 1-2 hours at room temperature. The starting current is about 100mA, and the current at the end of transfer is about 20mA.

[0261] Antibody incubation blocking After transfer, the membrane is rinsed with 1× Tris Buffered Saline (TBS). Block by incubation in 2% milk / TBST for 60 minutes at room temperature with shaking (e.g., on an orbital shaker or rocker). Wash several times with TBST.

[0262] Primary antibody incubation Incubate with primary antibody in the appropriate buffer for 1-2 hours at room temperature or overnight at 4°C. Wash five times with TBST for 5 minutes each.

[0263] Secondary antibody incubation Incubate secondary antibody in 2% milk / TBST for 1 hour at room temperature Wash five times with TBST for 5 minutes each.

[0264] Imaging After washing and during secondary antibody incubation, turn on the Fujifilm imaging system Combine ECL solution A and solution B in a 1:1 ratio, and use 1 mL of solution A + 1 mL of solution B for one membrane (dilute 1:1 with MilliQ water if necessary). Transfer the membrane to a clear plastic sheet protector Wipe off excess buffer with a paper towel Quickly add the ECL solution. The membrane is incubated with the ECL solution for 1 minute. Drain excess solution from the membrane and wipe off excess ECL reagent with a paper towel. Sandwich the membrane between two sheet protectors. Imaging is performed using a Fujifilm imager.

[0265] Example 6, Electroporation 3 mL of Optimizer complete medium containing 1000 IU / mL IL-2 is pre-warmed and equilibrated in each well of a 6-well plate. The gdT cells are centrifuged at 250 xg for 5 minutes at room temperature. Resuspend cells in Optimem medium to 10e6 cells / mL. Add 1 mL of cells at 10e6 cells / mL to 2 x 1.5 mL epi-tubes. Centrifuge the tube at 250 x g for 5 minutes at room temperature. Remove the supernatant completely, first with a P1000 pipette and then with a P200 pipette tip. Resuspend the cells in 100 µL of Optimem and pipette the cells up and down 3-5 times to break up the cell pellet. Add 15 μg of mRNA, mix, and place the cell / mRNA mixture into a 4 mm cuvette. Perform electroporation using the following settings: setting 500V square wave Pulse length 5ms 1 pulse 4mm cuvette Pulsing the cells Using a sterile, individually wrapped pipette, add approximately 500 μL of warmed media (from the 6-well plate) to the cuvette and carefully transfer the media / cells back to the 6-well plate. Place the cells in the incubator overnight.

[0266] Example 7, CD19 Cytotoxicity Assay This embodiment supports FIGS. 19 and 21.

[0267] Staining of target 697 cells Place 5e6 target cells into a 15 mL conical tube and spin down. The cells are washed twice with PBS to remove serum proteins. Prepare VPD450: add 1 µL of 1 mM VPD450 stock to 1 mL of PBS and mix. Resuspend cells in 500 μL of VPD450 / PBS. Unstained control cells are resuspended in PBS. Incubate in a 37°C water bath for 10 minutes. Add 9 mL of PBS and spin down the cells at 300 x g for 5 minutes. Resuspend the cells in 10 mL of PBS and spin down at 300 x g for 5 minutes. Cells are resuspended in 2 mL of complete Optimizer medium plus 1000 U / mL IL2. Viable cells are counted using trypan blue. Add OpTmizer+IL2 as needed to achieve a target cell concentration of 1e6 cells / mL. Incubate target and effector cells in a total volume of 200 µL in flow tubes according to the table below:

[0268] Preparation of gdT cells Collect the gdT cells and conditioned medium and place them in a 15 mL conical tube. Wash the flask with PBS and collect this in a separate 15 mL conical tube. Spin at 300 x g for 5 minutes at room temperature. Remove the conditioned medium from the cell pellet and transfer to a new conical tube. The conditioned medium is spun at 500 xg for 10 minutes to remove cell debris. The filtered conditioned medium is stored at -20°C. Resuspend and pool the cell pellets (from the cell pellet under conditioned medium and the PBS wash of the well). Resuspend in 300 μL of Optmizer (a volume small enough to ensure >5e6 cells / mL). Pipette up and down with a P1000 pipette to loosen cell clumps. Take 10 μL of cells and dilute 1:10 by adding to 90 μL of PBS. This 1:10 dilution of cells is counted with trypan blue. Add OpTmizer medium as needed to achieve a cell concentration of 5e6 cells / mL for each group. Target and effector cells are incubated in 48-well plates at 37° C. for 18 hours. Prior to Annexin V / 7AAD staining, assay samples are transferred from the wells to fully labeled flow tubes. Include a tube of unlabeled target cells in the wash steps. Wash the cells with 1 mL of flow buffer. Wash the cells once with 500 µL of Annexin V binding buffer. Resuspend unstained target cells in Annexin V binding buffer. Transfer half of the cells to a new 1.5 mL tube and heat sterilize in a 100°C heat block for 2 minutes. Cool the tube on ice and combine the dead and live cells. Use these cells for compensation. Resuspend in 100 µL of 1x annexin binding buffer containing 2.5 µL of annexin V-APC and 3 µL of 7AAD for 15 min at room temperature. Wash by adding 1 mL of Annexin V binding buffer per reaction. Spin down at 300 x g for 5 minutes. Decant and resuspend in 200 μL of Annexin V binding buffer (not FACS buffer!). A flow run is performed to determine the percentage of apoptotic and dead target cells by gating on VPD450 positive cells.

[0269] Example 8. Cytotoxicity Assay (HEL Cells and Kasumi Cells) This embodiment supports FIGS. 20 and 25.

[0270] VPD-labeled target cells (Kasumi1 cells or HEL cells) Place 5e6 target cells in a 15 mL conical tube and spin down. The cells are washed twice with PBS to remove serum proteins. Prepare VPD450: add 1 µL of 1 mM VPD450 stock to 1 mL of PBS and mix. Resuspend cells in 500 μL of VPD450 / PBS. Unstained control cells are resuspended in PBS. Incubate in a 37°C water bath for 10 minutes. Add 9 mL of PBS and spin down the cells at 300 x g for 5 minutes. Resuspend the cells in 10 mL of PBS and spin down at 300 x g for 5 minutes. Resuspend cells in 2 mL of complete Optimizer + 1000 U / mL IL2. Viable cells are counted using trypan blue. Add OpTmizer as needed to achieve a cell concentration of 1e6 cells / mL. Target and effector cells are incubated in a total volume of 200 μL in the flow tube.

[0271] Preparation of gdT cells Collect the gdT cells and conditioned medium from the multiwell plate and place them in a 15 mL conical tube. Wash the flask with PBS and collect this in a separate 15 mL conical tube. Spin at 300 x g for 5 minutes at room temperature. Resuspend in 150 μL of Optimizer medium. Pipette up and down with a P1000 pipette to loosen cell clumps. Take 10 μL of cells and add to 90 μL of PBS, diluting 1:10. This 1:10 dilution of cells is counted with trypan blue. Add OpTmizer medium as needed to achieve a cell concentration of 5e6 cells / mL for each group of gdT cells. Analyze cells by flow cytometry

[0272] This Example 9. shRNA Knockdown and Surface Expression of HLA Class I and Class II This embodiment supports FIGS. 32 and 33.

[0273] Day 0 - Transduce gdT cells: Thaw frozen gdT cells. Thaw the vial by transferring it directly to a -80°C to 37°C water bath. Transfer the contents of the cryovial to a 15 mL conical tube. Add 2 mL of thawing medium dropwise to the cells in the conical tube. Rinse the cryovial with 1 mL of thawing medium and add this rinse to the 15 mL conical tube, bringing the total volume in the conical tube to 4 mL. The cells are spun at 300 x g for 5 minutes at room temperature. Cells are resuspended in complete Optimizer medium + IL2 at 37°C to a cell density of 3e6 cells / mL. The cell suspension is placed in a flask of an appropriate size and placed in a TC incubator at 37°C. The cells are spun down in a 1.5 mL tube at 250 G for 5 minutes. Resuspend cells in unconcentrated lentivirus plus Optimizer medium. Cells are plated into wells of a 96-well plate for each condition and transduced overnight at 37°C / 5% CO2.

[0274] Day 1 - Medium change: Remove cells from the wells with a pipette The cells are spun down at 300g for 5 minutes and resuspended in 150 μL of complete Optimizer medium plus IL2. Cells are plated and incubated for 72 hours.

[0275] Day 4 - Preparing cells for flow cytometry: Spin the transduced gdT cells at 350 x g for 10 minutes. Decant the spun tubes and add 2.5 μL of anti-human B2m antibody + 97.5 μL of FACS mix to each tube and mix. The sample is allowed to stand at room temperature in a dark place for 10 minutes. After 10 minutes, add 3 μL of 7AAD to each sample and mix. The sample is returned to the dark and incubated at room temperature for 5 minutes. After 5 minutes, add 500 μL of FACS buffer to each sample and spin down at 300 g for 5 minutes. Resuspend the cells in 200 µL of FACS buffer and perform flow cytometry.

[0276] Example 10. Electroporation of Jurkat cells This embodiment supports FIG. The day before electroporation, split the cells to approximately 300K / mL. On the day of transfection, add 3 mL of medium to each required well of a 6-well plate. Collect cells in a 15mL conical tube and count - 15mL volume, 8.6e5 cells / mL = 12.9e6 cells Spin down at 200 x g for 10 minutes. Resuspend cells in 4.3 mL of Optimem (3e6 cells / mL). Add 1 mL of cell suspension to each 1.5 mL epi-tube. Spin down at 160 x g for 10 minutes. Resuspend the cells in 100 μL of Optimem. Add 5 μL (= 5 μg) of GFP mRNA to the tube. Mix by pipetting up and down several times and transfer the contents to a cuvette. Perform electroporation using the following parameters: Voltage: 140V Pulse length: 5ms Number of pulses: 2 Pulse interval: 0.1 seconds Cuvette spacing: 2mm Transfer the cells to a 6-well plate. Culture overnight in a TC incubator Approximately 24 hours after transfection, cells are analyzed by flow cytometry.

[0277] Sequence Product List In the following sections, the sequence listing is followed by a description of each sequence. For each, the specification provides first the region representing the signal peptide and its residues, then the target scFv (hereinafter used as an abbreviation for the residues of the sequence that constitutes the scFv portion of the target scFv) and its residues, the target VL (hereinafter used as an abbreviation for the residues of the sequence that constitutes the VL portion of the target scFv) and its residues, the target linker (hereinafter used as an abbreviation for the residues of the sequence that constitutes the linker portion) and its residues, the target VH (hereinafter also used as an abbreviation for the residues of the sequence that constitutes the VH of the target scFv) and its residues, the central linker (an abbreviation for the flexible linker in the specification; see, for example, Figure 1 ) and its residues, the gdT scFv, the gdT VH, the gdT linker, and the gdT VL. Also provided herein are free energies, gdT CAIs, and ORF numbers, as appropriate.

[0278] [Sequence table] SEQ ID NO: 1 is the sequence name of IL2 CD19 CD3 STAR. It is the complete STAR construct. It is the amino acid sequence. The sequence is: MYRMQLLSCIALSLALVTNSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEI KGGGGSGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPDGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWG QGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0279] SEQ ID NO: 1 includes signal peptide: 1-20; target scFv: 21-270; target VL: 21-131; target linker: 132-146; target VH: 147-270; central linker: 271-275; gdT scFv: 276-518; gdT VH: 397-410; gdT linker: 397-410; gdT VL: 411-518.

[0280] SEQ ID NO: 2 is the sequence name of IL2 CD19 CD3 STAR. It is the complete STAR construct. It is the amino acid sequence. The sequence is:

[0281] SEQ ID NO: 2 includes signal peptide: 1-60; target scFv: 61-810; target VL: 61-393; target linker: 394-438; target VH: 439-810; central linker: 811-825; gdT scFv: 826-1544; gdTVH: 1189-1230; gdT linker: 1189-1230; gdTVL: 1231-1544.

[0282] SEQ ID NO: 3 is the sequence name of modified serum albumin (mSA). It is a signal peptide construct. It is an amino acid sequence. The sequence is: MKWVTFISLLFLFSSSSRA.

[0283] SEQ ID NO: 3 includes signal peptides 1-19.

[0284] SEQ ID NO: 4 is the sequence name of human stem cell factor (hSCF). It is a signal peptide construct. It is an amino acid sequence. The sequence is: MKKTQTWILTCIYLQLLLFNPLVKT.

[0285] SEQ ID NO: 4 includes signal peptides 1-25.

[0286] SEQ ID NO: 5 is the sequence name of modified serum albumin (mSA). It is a signal peptide construct. It is a DNA sequence. The sequence is: ATGAAATGGGTTACTTTTATTAGTTTATTATTCCTGTTCAGCTCCAGCTCCAGGGCC.

[0287] SEQ ID NO: 5 includes signal peptides 1-57.

[0288] SEQ ID NO: 6 is the sequence name of human stem cell factor (hSCF). It is a signal peptide construct. It is a DNA sequence. The sequence is: ATGAAGAAAACTCAAACTTGGATACTAACTTGCATCTACCTGCAGCTGCTGCTCTTCAACCCCTTGGTGAAGACG.

[0289] SEQ ID NO: 6 includes signal peptides 1-75.

[0290] SEQ ID NO: 7 is the sequence name of CD19 scFv ECOg(154). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GACATCCAGCTCACCCAGAGCCCCGCATCCCTGGCCGTGTCCCTGGGGCAGCGCGCAACCATCTCCTGCAAGGCTTCCCAGTCCGTGGACTACGACGGGGACTCCTACCTGAACTGGTACCAGCAGATCCCCGGGCAGCCACCGAAGCTGCTGATCTACGACGCCTCCAACCTGGTGTCCGGGATTCCTCCGCGGTTCTCCGGGAGCGGGTCCGGGACCGACTTCACCCTGAACATCCATCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGTCCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAGGTGGCGGAGGCTCCGGTGGCGGAGGTTCCGGTGGCGGCGGCTCCCAGGTGCAGCTGCAGCAGTCAGGGGCCGAGCTGGTGAGGCCCGGGAGCTCCGTGAAGATCTCCTGCAAGGCCTCCGGGTACGCCTTCTCCTCCTACTGGATGAACTGGGTGAAGCAGAGGCCCGGGCAGGGGCTGGAGTGGATCGGGCAGATCTGGCCCGGGGACGGGGACACCAACTACAACGGGAAGTTCAAGGGGAAGGCTACCCTCACCGCCGACGAGAGCTCCTCCACCGCCTACATGCAGCTGAGCTCCCTCGCCTCCGAGGACTCCGCCGTGTACTTCTGCGCCCGGCGCGAGACCACCACCGTGGGGCGCTACTACTACGCCATGGACTACTGGGGCCAGGGGACCACCGTGACCGTGAGCTCC is as follows.

[0291] SEQ ID NO: 7 includes target scFv: 1 to 750; target VL: 1 to 333; target linker: 334 to 378; target VH: 379 to 750; free energy: -345.2; gdT CAI: 0.89467859593316; number of ORFs: 1.

[0292] SEQ ID NO: 8 is the sequence name of CD19 scFv ECOg(94). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0293] SEQ ID NO: 8 includes target scFv: 1 to 750; target VL: 1 to 333; target linker: 334 to 378; target VH: 379 to 750; free energy: -360.7; gdT CAI: 0.767359962199379; number of ORFs: 2.

[0294] Array number 9 is the sequence name of CD19 scFv ECOg(139). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GACATCCAGCTGACGCAGTCGCCCGCGTCGCTCGCCGTGTCGCTCGGGCAACGCGCGACGATCTCGTGCAAGGCGTCGCAGTCCGTCGACTACGACGGCGACTCGTACCTGAACTGGTACCAGCAGATCCCCGGGCAGCCGCCGAAGCTGCTGATCTACGACGCGAGCAACCTCGTGTCCGGCATTCCGCCGCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACGCTGAACATTCACCCCGTCGAGAAGGTCGACGCCGCGACGTACCACTGCCAGCAGTCCACCGAGGACCCGTGGACGTTCGGCGGCGGCACGAAGCTGGAGATCAAAGGTGGCGGCGGTTCCGGTGGCGGTGGTTCCGGTGGCGGCGGCTCGCAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGCGACCCGGGTCGTCCGTGAAGATCTCGTGCAAGGCGTCCGGGTACGCATTCTCGTCGTACTGGATGAACTGGGTGAAGCAGCGACCCGGGCAGGGGCTGGAGTGGATCGGGCAGATCTGGCCCGGCGACGGCGACACGAACTACAACGGCAAGTTCAAGGGCAAGGCGACGCTGACCGCCGACGAGTCGTCGTCCACCGCGTACATGCAGCTGTCGTCGCTCGCGAGCGAGGACAGCGCCGTGTACTTCTGCGCGCGGCGCGAGACGACGACCGTCGGGCGCTACTACTACGCGATGGACTACTGGGGCCAGGGCACGACCGTGACCGTGTCCAGC

[0295] SEQ ID NO: 9 includes target scFv: 1 to 750; target VL: 1 to 333; target linker: 334 to 378; target VH: 379 to 750; free energy: -357.7; gdT CAI: 0.724573751544555; number of ORFs: 1.

[0296] SEQ ID NO: 10 is the sequence name of CD19 scFv ECOg(195). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0297] SEQ ID NO: 10 includes target scFv: 1 to 750; target VL: 1 to 333; target linker: 334 to 378; target VH: 379 to 750; free energy: -356; gdT CAI: 0.858340915819879; and number of ORFs: 2.

[0298] SEQ ID NO: 11 is the sequence name of CD19 scFv ECOg(160). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GACATCCAGCTGACCCAGTCACCCGCTAGCCTGGCCGTGTCCCTGGGCCAGCGAGCCACGATCTCCTGCAAGGCCAGCCAGTCCGTGGACTACGACGGGGACTCCTACCTCAACTGGTACCAGCAGATCCCCGGCCAGCCACCGAAGCTGCTGATCTACGACGCCTCCAACCTGGTGAGCGGGATTCCGCCGCGGTTCAGCGGGTCCGGGTCCGGGACCGACTTCACCCTCAACATCCATCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGTCCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAGGCGGTGGTGGCTCCGGAGGCGGTGGCTCTGGTGGCGGCGGCTCCCAGGTGCAGCTCCAGCAGAGCGGGGCTGAGCTGGTGAGGCCCGGGTCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGGTACGCCTTCTCCAGCTACTGGATGAACTGGGTGAAGCAGAGGCCCGGGCAGGGGCTGGAGTGGATCGGGCAGATCTGGCCCGGGGACGGGGACACCAACTACAACGGGAAGTTCAAGGGGAAGGCCACGCTGACCGCCGACGAGAGCAGCTCCACCGCCTACATGCAGCTGTCCAGCCTGGCCTCCGAGGACTCCGCCGTGTACTTCTGCGCCCGGCGCGAGACCACCACCGTGGGGCGCTACTACTACGCCATGGACTACTGGGGCCAGGGGACCACCGTGACCGTGTCCAGC is as follows.

[0299] SEQ ID NO: 11 includes target scFv: 1 - 750; target VL: 1 - 333; target linker: 334 - 378; target VH: 379 - 750; free energy: -354.8; gdT CAI: 0.876843948859594; number of ORFs: 2.

[0300] SEQ ID NO: 12 is the sequence name of CD3 scFv ECOg(70). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: is.

[0301] SEQ ID NO: 12 includes gdT scFv: 1 to 729; gdT VH: 364 to 405; gdT linker: 364 to 405; gdT VL: 406 to 729; free energy: −320.1; gdT CAI: 0.924743; and number of ORFs: 1.

[0302] SEQ ID NO: 13 is the sequence name of CD3 scFv ECOg(197). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: is.

[0303] SEQ ID NO: 13 includes gdT scFv: 1 to 729; gdT VH: 364 to 405; gdT linker: 364 to 405; gdT VL: 406 to 729; free energy: -341.1; gdT CAI: 0.803206301402276; number of ORFs: 2.

[0304] SEQ ID NO: 14 is the sequence name of CD3 scFv ECOg(109). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: is.

[0305] SEQ ID NO: 14 includes gdT scFv: 1 to 729; gdT VH: 364 to 405; gdT linker: 364 to 405; gdT VL: 406 to 729; free energy: −334.7; gdT CAI: 0.70167404462703; and number of ORFs: 1.

[0306] SEQ ID NO: 15 is the sequence name of CD3 scFv ECOg(119). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: is.

[0307] SEQ ID NO: 15 includes gdT scFv: 1 to 729; gdT VH: 364 to 405; gdT linker: 364 to 405; gdT VL: 406 to 729; free energy: −328.1; gdT CAI: 0.739432850740138; number of ORFs: 0.

[0308] Accession number 16 is the sequence name of CD3 scFv ECOg(45). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: GACATCAAGCTGCAGCAGTCCGGAGCCGAGTTGGCACGGCCCGGGGCCTCCGTGAAGATGTCCTGCAAGACGTCCGGGTACACGTTCACCCGGTACACCATGCACTGGGTGAAGCAGCGGCCCGGGCAGGGGCTGGAATGGATCGGGTACATCAACCCCAGCCGCGGGTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACGCTGACCACCGACAAGTCGTCGTCGACCGCCTACATGCAGCTGAGCTCGCTGACCAGCGAGGACAGCGCCGTCTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGGACCACGCTGACAGTGAGCAGCGTGGAAGGCGGCTCCGGCGGCTCCGGAGGTTCCGGAGGCAGCGGCGGCGTCGACGACATCCAGCTCACCCAGTCCCCGGCCATCATGTCGGCGAGCCCCGGCGAGAAGGTGACCATGACGTGCCGGGCCTCCAGCTCGGTGTCCTACATGAACTGGTACCAGCAGAAGTCGGGGACCAGCCCCAAGCGGTGGATCTACGACACCAGCAAGGTGGCCAGCGGGGTCCCCTACCGCTTCTCGGGGTCCGGGTCCGGGACCTCGTACTCGCTGACCATCTCCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGTCCTCGAACCCGCTCACCTTCGGGGCCGGCACCAAGCTGGAGCTGAAG

[0309] SEQ ID NO: 16 includes gdT scFv: 1 to 729; gdT VH: 364 to 405; gdT linker: 364 to 405; gdT VL: 406 to 729; free energy: -323.7; gdT CAI: 0.817020513172323; number of ORFs: 0.

[0310] SEQ ID NO: 17 is the sequence name of the PTK7 scFv. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSTYLMYWVRQAPGKTLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPMYTFGQGTKLEIK.

[0311] SEQ ID NO: 17 includes target scFv: 1 to 236; target VL: 128 to 236; target linker: 113 to 127; and target VH: 1 to 236.

[0312] SEQ ID NO: 18 is the sequence name of the PTK7 scFv. It is a tumor-targeting scF construct. It is a DNA sequence. The sequence is: is.

[0313] SEQ ID NO: 18 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -268.3; gdT CAI: 0.722372746155158; number of ORFs: 6.

[0314] SEQ ID NO: 19 is the sequence name of PTK7 ECOg(74). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0315] SEQ ID NO: 19 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -336.8; gdT CAI: 0.861158; number of ORFs: 0.

[0316] SEQ ID NO: 20 is the sequence name of PTK7 ECOg(18). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0317] SEQ ID NO: 20 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -356; gdT CAI: 0.803927502492027; and number of ORFs: 4.

[0318] SEQ ID NO: 21 is the sequence name of PTK7 ECOg(70). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0319] SEQ ID NO: 21 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -355.6; gdT CAI: 0.830356865758289; number of ORFs: 3.

[0320] SEQ ID NO: 22 is the sequence name of PTK7 ECOg(68). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0321] SEQ ID NO: 22 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -355.5; gdT CAI: 0.811107619741586; number of ORFs: 2.

[0322] SEQ ID NO: 23 is the sequence name of PTK7 ECOg(2). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0323] SEQ ID NO: 23 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -350.2; gdT CAI: 0.803914913406133; number of ORFs: 2.

[0324] SEQ ID NO: 24 is the sequence name of the hSCF ligand. It is a tumor targeting ligand construct. It is an amino acid sequence. The sequence is: EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSS.

[0325] SEQ ID NO: 24 includes target ligands: 1-142.

[0326] SEQ ID NO: 25 is the sequence name of the hSCF ligand. It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: GAGGGGATCTGCAGGAACAGGGTGACCAACAATGTGAAGGATGTGACCAAGCTGGTGGCCAACCTGCCCAAGGACTACATGATCACCCTGAAGTATGTGCCAGGGATGGATGTGCTGCCCAGCCACTGCTGGATCTCTGAGATGGTGGTGCAGCTGTCTGACTCCCTGACAGACCTGCTGGACAAGTTCTCCAACATCTCAGAGGGCTGTCCAA CTACTCCATCATTGACAAGCTGGTGAACATAGTGGATGACCTGGTGGAGTGTGTGAAGGAGAACTCCTCCAAGGACCTGAAGAAGTCCTTCAAGTCCCCTGAGCCCAGGCTGTTCACCCCTGAGGAGTTCTTCAGGATCTTCAACAGGTCCATTGATGCCTTCAAGGACTTTGTGGTGGCCTCTGAGACCTCTGACTGTGTGGTGTCCTCA.

[0327] SEQ ID NO: 25 includes target ligand: 1-426; free energy: -157.8; gdT CAI: 0.937866445503855; number of ORFs: 3.

[0328] SEQ ID NO: 26 is the sequence name of the hSCF ligand ECOg(87). This is a tumor-targeting ligand construct. This is a DNA sequence. The sequence is: GAGGGGATCTGCCGGAACCGGGTGACCAACAACGTGAAGGACGTGACCAAGCTGGTGGCCAACCTGCCCAAGGACTACATGATCACCCTGAAGTACGTGCCCGGGATGGACGTGCTGCCCAGCCACTGCTGGATCAGCGAGATGGTGGTGCAGCTGTCCGACTCCCTGACCGACCTGCTGGACAAGTTCTCCAACATCAGCGAGGGGCTGAGCAA CTACTCCATCATCGACAAGCTGGTGAACATCGTGGACGACCTGGTGGAGTGCGTGAAGGAGAACAGCAGCAAGGACCTGAAGAAGTCCTTCAAGAGCCCCGAGCCCCGGCTGTTCACGCCCGAGGAGTTCTTCCGGATCTTCAACCGGAGCATCGACGCCTTCAAGGACTTCGTGGTGGCCAGCGAGACCAGCGACTGCGTGGTGTCCTCC.

[0329] SEQ ID NO: 26 includes target ligand: 1-426; free energy: -160.7; gdT CAI: 0.928424; number of ORFs: 0.

[0330] SEQ ID NO: 27 is the sequence name of the hSCF ligand ECOg(62). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: GAGGGGATCTGCCGGAACCGGGTGACCAACAATGTGAAGGACGTCACAAAGTTGGTCGCCAATCTGCCGAAGGACTACATGATTACGCTGAAGTACGTCCCCGGAATGGATGTGCTGCCCAGCCACTGCTGGATTTCGGAGATGGTGGTGCAGCTGTCCGACAGTCTGACCGATCTGCTGGACAAGTTCAGCAACATCTCCGAAGGGCTGTCCAA CTACAGCATCATCGATAAGCTGGTCAACATCGTCGACGATCTGGTGGAGTGCGTCAAAGAGAACAGCAGCAAAGATCTGAAGAAGTCGTTCAAATCGCCGGAGCCGCGGCTGTTCACACCGGAGGAGTTCTTCCGGATCTTCAATCGGTCGATCGACGCCTTCAAAGATTTTGTGGTGGCCAGCGAAACCAGCGACTGCGTCGTCAGCAGC.

[0331] SEQ ID NO: 27 includes target ligand: 1-426; free energy: -164.6; gdT CAI: 0.805174371566857; number of ORFs: 2.

[0332] SEQ ID NO: 28 is the sequence name of the hSCF ligand ECOg(61). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: GAGGGGATCTGCCGGAACCGCGTGACGAACAACGTGAAGGACGTCACGAAGCTCGTCGCGAACCTGCCGAAGGACTACATGATCACCCTGAAGTACGTCCCCGGATGGACGTGCTCCCCTCGCACTGCTGGATCTCCGAGATGGTCGTCCAGCTGTCCGACTCCCTGACGACCTCCTCGACAAGTTCTCCAACATCTCCGAGGGGCTCTCCAA CTACTCGATCATCGACAAGCTGGTGAACATCGTGGACGACCTCGTGGAGTGCGTCAAGGAGAACTCCTCGAAGGACCTCAAGAAGAGCTTCAAGTCGCCCGAGCCGCGGCTCTTCACGCCCGAGGAGTTCTTCCGGATCTTCAACCGGAGCATCGACGCCTTCAAGGACTTCGTGGTGGCCTCCGAGACGTCCGACTGCGTCGTGTCGAGC.

[0333] SEQ ID NO: 28 includes target ligand: 1-426; free energy: -163.9; gdT CAI: 0.83416441300645; number of ORFs: 0.

[0334] SEQ ID NO: 29 is the sequence name of the hSCF ligand ECOg(2). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: GAGGGGATCTGCCGGAACCGGGTGACCAACAACGTGAAGGACGTCACCAAGCTGGTCGCCAACCTGCCCAAGGACTACATGATCACCTTGAAGTACGTGCCCGGCATGGACGTCCTGCCCAGCCACTGCTGGATCTCCGAGATGGTCGTCCAGCTCAGCGACTCCCTGACCGACCTCCTCGACAAGTTCTCCAACATCTCCGAGGGGCTCAGCAA CTACTCCATCATCGACAAGCTCGTGAACATAGTGGATGACCTCGTGGAGTGCGTGAAGGAGAACAGCTCCAAGGACTTGAAGAAGTCCTTCAAGTCCCCGGAGCCCAGGCTGTTCACGCCCGAGGAGTTCTTCAGGATCTTCAACCGATCCATTGACGCCTTCAAGGACTTCGTGGTGGCCTCCGAGACCAGCGACTGCGTGGTGTCCTCC.

[0335] SEQ ID NO: 29 includes target ligand: 1-426; free energy: -163.7; gdT CAI: 0.886419970682739; number of ORFs: 2.

[0336] SEQ ID NO: 30 is the sequence name of the hSCF ligand ECOg(48). This is a tumor-targeting ligand construct. This is a DNA sequence. The sequence is: GAGGGGATCTGCCGCAACCGCGTGACGAACAACGTGAAGGACGTGACGAAGCTCGTGGCGAACCTGCCCAAGGACTACATGATCACCCTGAAGTACGTCCCCGGATGGACGTGCTGCCCAGCCACTGCTGGATCTCCGAGATGGTGGTGCAGCTGAGCGACAGCCTGACGGACCTGCTGGACAAGTTCAGCAACATCTCCGAGGGGCTGAGCAA CTACAGCATCATCGACAAGCTGGTGAACATCGTGGACGACCTGGTGGAGTGCGTGAAGGAGAACAGCTCCAAGGACCTGAAGAAGAGCTTCAAGTCGCCCGAGCCCCGGCTGTTCACGCCCGAGGAGTTCTTCCGGATCTTCAACCGGAGCATCGACGCCTTCAAGGACTTCGTCGTGGCGAGCGAGACGTCCGACTGCGTCGTGTCCTCC.

[0337] SEQ ID NO: 30 includes target ligand: 1-426; free energy: -163.5; gdT CAI: 0.876702550243938; number of ORFs: 0.

[0338] SEQ ID NO: 31 is the sequence name of albumin. It is a fusion site construct. It is an amino acid sequence. The sequence is: is.

[0339] SEQ ID NO: 31 includes fusion sites: 1-590.

[0340] SEQ ID NO: 32 is the sequence name of albumin. It is a fusion site construct. It is a DNA sequence. The sequence is:

[0341] SEQ ID NO: 32 includes fusion site: 1 to 1770, free energy: -557.6; gdT CAI: 0.787786351918606; number of ORFs: 21.

[0342] SEQ ID NO: 33 is the sequence name of albumin ECOg(8). It is a fusion site construct. It is a DNA sequence. The sequence is:

[0343] SEQ ID NO: 33 includes fusion site: 1-1770; free energy: -755.4; gdT CAI: 0.932865573516391; number of ORFs: 0.

[0344] SEQ ID NO: 34 is the sequence name of albumin ECOg(60). It is a fusion site construct. It is a DNA sequence. The sequence is:

[0345] SEQ ID NO: 34 includes fusion site: 1-1770; free energy: -753.1; gdT CAI: 0.893692649517376; number of ORFs: 0.

[0346] SEQ ID NO: 35 is the sequence name of albumin ECOg(91). It is a fusion site construct. It is a DNA sequence. The sequence is:

[0347] SEQ ID NO: 35 includes fusion site: 1-1770; free energy: -750.9; gdT CAI: 0.909026600521571; number of ORFs: 7.

[0348] SEQ ID NO: 36 is the sequence name of albumin ECOg(51). It is a fusion site construct. It is a DNA sequence. The sequence is:

[0349] SEQ ID NO: 36 includes fusion site: 1-1770; free energy: -749.1; gdT CAI: 0.879685715389261; number of ORFs: 0.

[0350] SEQ ID NO: 37 is the sequence name of albumin ECOg(62). It is a fusion site construct. It is a DNA sequence. The sequence is:

[0351] SEQ ID NO: 37 includes fusion site: 1-1770; free energy: -745.2; gdT CAI: 0.955655916201255; number of ORFs: 4.

[0352] SEQ ID NO: 38 is the sequence name of GD2 scFv. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMKYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRAD.

[0353] SEQ ID NO: 38 includes target scFv: 1 to 244; target VL: 129 to 244; target linker: 114 to 128; and target VH: 1 to 113.

[0354] SEQ ID NO: 39 is the sequence name of the GD2 scFv. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0355] SEQ ID NO: 39 includes target scFv: 1 to 732; target VL: 385 to 732; target linker: 340 to 384; target VH: 1 to 339; free energy: -243.8; gdT CAI: 0.70651634854966; number of ORFs: 9.

[0356] SEQ ID NO: 40 is the sequence name of GD2 scFv ECOg(18). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0357] SEQ ID NO: 40 includes target scFv: 1 to 732; target VL: 385 to 732; target linker: 340 to 384; target VH: 1 to 339; free energy: -328.1; gdT CAI: 0.877232; number of ORFs: 0.

[0358] SEQ ID NO: 41 is the sequence name of GD2 scFv ECOg(84). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GAGGTCCAGCTGCTGCAGTCGGGGCCCGAGCTGGAGAAGCCCGGGGCCTCCGTCATGATCTCGTGCAAGGCCTCCGGGTCCTCGTTCACCGGGTACAACATGAACTGGGTGCGCCAGAACATCGGGAAGTCGCTGGAGTGGATCGGGGCCATCGACCCCTACTACGGCGGCACCAGCTACAACCAGAAGTTCAAGGGGCGCGCCACGCTGACGGTGGACAAGTCGTCGTCGACCGCCTACATGCACCTGAAGTCGCTGACGTCGGAGGACTCCGCCGTCTACTACTGCGTCAGCGGGATGAAGTACTGGGGCCAGGGGACCTCGGTCACCGTGTCCTCCGGCGGCGGAGGAAGCGGAGGAGGAGGCTCCGGCGGAGGAGGCTCCGACGTCGTGATGACGCAGACCCCGCTGTCGCTCCCGGTGTCCCTCGGGGACCAGGCCTCCATCAGCTGCCGGAGCTCGCAGTCCCTGGTGCACCGGAACGGGAACACCTACCTCCACTGGTACCTGCAGAAGCCGGGGCAGTCGCCCAAGCTGCTGATCCACAAGGTGTCCAACAGGTTCTCCGGGGTCCCCGACCGCTTCAGCGGGAGCGGGAGCGGGACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACCTCGGGGTCTACTTCTGCAGCCAGAGCACCCACGTGCCACCGCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTCAAGCGGGCCGAC.

[0359] SEQ ID NO: 41 includes target scFv: 1-732; target VL: 385-732; target linker: 340-384; target VH: 1-339; free energy: -351.8; gdT CAI: 0.7977343893656; number of ORFs: 0.

[0360] SEQ ID NO: 42 is the sequence name of GD2 scFv ECOg(88). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0361] SEQ ID NO: 42 includes target scFv: 1 to 732; target VL: 385 to 732; target linker: 340 to 384; target VH: 1 to 339; free energy: -340; gdT CAI: 0.799370908165938; and number of ORFs: 1.

[0362] SEQ ID NO: 43 is the sequence name of GD2 scFv ECOg(2). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0363] SEQ ID NO: 43 includes target scFv: 1 to 732; target VL: 385 to 732; target linker: 340 to 384; target VH: 1 to 339; free energy: -339.9; gdT CAI: 0.78785010580307; number of ORFs: 2.

[0364] SEQ ID NO: 44 is the sequence name of GD2 scFv ECOg(86). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0365] SEQ ID NO: 44 includes target scFv: 1 to 732; target VL: 385 to 732; target linker: 340 to 384; target VH: 1 to 339; free energy: -336.5; gdT CAI: 0.849533769168508; number of ORFs: 0.

[0366] SEQ ID NO: 45 is the sequence name of Integrin aVb3 scFv. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLEESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQIPEKRLEWVAKVSSGGGSTYYLDTVQGRFTISRDNAKNTLYLQMSSLNSEDTAMYYCARHNYGSFAYWGQGTLVTVSAAKGGGGSGGGGSGGGGSELVMTQTPATLSVTPGDSVSLSCRASQSISNHLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPHTFGGGTKLEIK.

[0367] SEQ ID NO: 45 includes target scFv: 1 to 241; target VL: 135 to 241; target linker: 120 to 134; target VH: 1 to 119.

[0368] SEQ ID NO: 46 is the sequence name of Integrin aVb3 scFv. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0369] SEQ ID NO: 46 includes target scFv: 1 to 723; target VL: 403 to 723; target linker: 358 to 402; target VH: 1 to 357; free energy: -326.8; gdT CAI: 0.836939021276497; number of ORFs: 8.

[0370] SEQ ID NO: 47 is the sequence name of Integrin aVb3 scFv ECOg(62). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0371] SEQ ID NO: 47 includes target scFv: 1 to 723; target VL: 403 to 723; target linker: 358 to 402; target VH: 1 to 357; free energy: -324.4; gdT CAI: 0.868908; and number of ORFs: 1.

[0372] SEQ ID NO: 48 is the sequence name of Integrin aVb3 scFv ECOg(26). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GAAGTCCAGCTGGAGGAGAGCGGCGGTGGCCTCGTGAAGCCCGGCGGTTCGCTCAAGCTGAGCTGCGCGGCCAGCGGGTTCGCCTTCAGCTCGTACGACATGAGCTGGGTGCGGCAGATCCCCGAGAAGCGCCTGGAGTGGGTCGCCAAGGTGTCCTCCGGCGGCGGCTCGACGTACTACCTGGACACCGTGCAGGGGCGCTTCACGATCAGCCGGGACAACGCGAAGAACACCCTGTACCTCCAGATGTCCTCGCTGAACTCCGAGGACACCGCGATGTACTACTGCGCGCGGCACAACTACGGGAGCTTCGCCTACTGGGGCCAGGGCACCCTGGTCACCGTGAGCGCGGCGAAAGGCGGTGGCGGCAGTGGTGGCGGAGGCTCTGGCGGCGGTGGCTCCGAGCTGGTGATGACCCAGACCCCGGCGACGCTCTCCGTGACGCCCGGGGACTCCGTGAGCCTGTCCTGCCGGGCCTCGCAGAGCATCTCCAACCACCTGCACTGGTACCAGCAGAAGAGCCACGAGTCGCCCCGGCTGCTCATCAAGTACGCCTCGCAGAGCATCTCCGGGATTCCCTCGCGGTTCTCCGGGAGCGGCTCGGGGACGGACTTCACGCTGTCCATCAACTCCGTGGAGACCGAGGACTTCGGGATGTACTTCTGCCAGCAGAGCAACTCCTGGCCGCACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG.

[0373] SEQ ID NO: 48 includes target scFv: 1 - 723; target VL: 403 - 723; target linker: 358 - 402; target VH: 1 - 357; free energy: -342.6; gdT CAI: 0.773341369143768; number of ORFs: 1.

[0374] SEQ ID NO: 49 is the sequence name of Integrin aVb3 scFv ECOg(12). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0375] SEQ ID NO: 49 includes target scFv: 1 to 723; target VL: 403 to 723; target linker: 358 to 402; target VH: 1 to 357; free energy: -339.8; gdT CAI: 0.762733907249084; number of ORFs: 2.

[0376] SEQ ID NO: 50 is the sequence name of Integrin aVb3 scFv ECOg(48). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0377] SEQ ID NO: 50 includes target scFv: 1 to 723; target VL: 403 to 723; target linker: 358 to 402; target VH: 1 to 357; free energy: -329.4; gdT CAI: 0.82265589760209; number of ORFs: 0.

[0378] SEQ ID NO: 51 is the sequence name of Integrin aVb3 scFv ECOg(97). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0379] SEQ ID NO: 51 includes target scFv: 1 to 723; target VL: 403 to 723; target linker: 358 to 402; target VH: 1 to 357; free energy: -329.3; gdT CAI: 0.777965026449684; number of ORFs: 0.

[0380] SEQ ID NO: 52 is the sequence name of the SSTR2 scFv. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRNRKNYLAWYQQKPDQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWFRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTLVTVS.

[0381] SEQ ID NO: 52 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0382] SEQ ID NO: 53 is the sequence name of the SSTR2 scFv. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GACATTGTGATGACCCAGAGCCCAGACTCCCTGGCTGTGAGCCTAGGGGAGAGGGCCACCATCAACTGCAAGTCCTCTCAGAGCCTCCTCAACTCCAGGAACAGGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCAGACCAGAGCCCCAAGCTGCTCATCTACTGGGCCTCCACCAGGGAGTCTGGGGTGCCTGACAGGTTCTCTGGGTCTGGGTCTGGGACTGACTTCACCCTGACCATCAGCTCCCTGCAGGCTGAGGATGTGGCTGTGTACTACTGCAAGCAGAGCTACTACCTGTGGACCTTTGGTGGAGGCACCAAGGTGGAGATCAAAGGAGGTGGAGGCTCTGGTGGTGGAGGCTCTGGTGGTGGAGGCTCTGAGGTGCAGCTGGTGGAGTCTGGTGGTGGCCTGGTCCAGCCAGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCTGGGTTCACCTTCTCAGACTATGGGATGGCCTGGTTCAGGCAGGCCCCAGGGAAGGGCCTGGAGTGGGTGAGCTTCATCTCCAACCTGGGCTACTCCATCTACTATGCTGACTCTGTGAAGGGCAGGTTCACCATCAGCAGGGACAATGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACAGCTGTGTACTACTGTGCCAGGGCCCCATATGACTATGACAGCTTTGACCCCATGGACTACTGGGGCCAGGGGACCCTGGTGACTGTGAGC.

[0383] SEQ ID NO: 53 includes target scFv: 1-744; target VL: 1-336; target linker: 337-381; target VH: 382-744; free energy: -344; gdT CAI: 0.841055073258909; number of ORFs: 7.

[0384] SEQ ID NO: 54 is the sequence name of SSTR2 svFv ECOg(72). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0385] SEQ ID NO: 54 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -325.1; gdT CAI: 0.865568; number of ORFs: 1.

[0386] SEQ ID NO: 55 is the sequence name of SSTR2 svFv ECOg(64). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0387] SEQ ID NO: 55 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -333.3; gdT CAI: 0.782038165701074; number of ORFs: 4.

[0388] Array number 56 is the sequence name of SSTR2 svFv ECOg(15). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GACATCGTCATGACCCAGTCACCTGACAGCCTGGCCGTCAGCCTGGGCGAACGGGCCACCATCAACTGTAAGTCATCTCAGAGCCTGCTGAACAGCCGGAACCGGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCTGATCAGTCACCTAAGCTGCTGATCTACTGGGCCTCAACCAGAGAGTCCGGCGTGCCTGACAGGTTCAGCGGGTCCGGGTCCGGGACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTCTATTACTGTAAGCAGTCTTATTACCTGTGGACCTTCGGCGGCGGCACCAAGGTCGAGATCAAAGGCGGCGGCGGCTCAGGTGGTGGTGGCTCCGGCGGCGGTGGCTCTGAGGTCCAGCTGGTCGAGTCCGGCGGTGGCCTGGTCCAGCCAGGCGGCAGCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCTGACTACGGCATGGCCTGGTTCAGACAGGCCCCAGGCAAGGGCCTGGAGTGGGTCAGCTTCATCAGCAACCTGGGCTACAGCATCTATTACGCCGACAGCGTCAAGGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACGGCCGTCTATTACTGCGCCCGGGCCCCTTATGACTATGACAGCTTTGACCCCATGGACTACTGGGGCCAGGGGACCCTGGTGACCGTGTCA.

[0389] SEQ ID NO: 56 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -331.6; gdT CAI: 0.812422023876491; number of ORFs: 8.

[0390] SEQ ID NO: 57 is the sequence name of SSTR2 svFv ECOg(50). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0391] SEQ ID NO: 57 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -328.9; gdT CAI: 0.878055710303915; number of ORFs: 3.

[0392] SEQ ID NO: 58 is the sequence name of SSTR2 svFv ECOg(42). It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: GATATCGTGATGACCCAGTCCCCGGACTCCCTGGCAGTGTCCCTCGGGGAGCGGGCCACCATCAACTGCAAGAGCTCCCAGTCCCTGCTGAACTCCCGGAACCGGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGACCAGTCCCCGAAGCTGCTGATCTACTGGGCCAGCACCCGGGAATCCGGGGTGCCCGACCGCTTCTCCGGGTCCGGGTCCGGGACCGACTTCACCCTGACCATCAGCTCCCTGCAGGCCGAGGACGTGGCAGTGTACTACTGCAAGCAGTCCTACTACCTGTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAAGGCGGCGGCGGCTCTGGAGGAGGAGGCTCCGGCGGCGGAGGTTCCGAGGTGCAGCTGGTGGAGTCCGGCGGAGGACTGGTGCAGCCCGGCGGCTCCCTGCGACTGTCCTGCGCCGCCTCCGGGTTCACCTTCTCCGACTACGGGATGGCCTGGTTCCGGCAGGCTCCCGGGAAGGGGCTGGAGTGGGTGTCCTTCATCTCCAACCTGGGGTACTCCATCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCAGTGTACTACTGCGCCCGGGCCCCGTACGACTACGACTCCTTCGACCCCATGGACTACTGGGGCCAGGGGACCCTGGTGACCGTGTCC is as follows.

[0393] SEQ ID NO: 58 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -328.6; gdT CAI: 0.886975497635559; number of ORFs: 1.

[0394] SEQ ID NO: 59 is the sequence name of the 2xSST28 3xg4S ligand. It is a tumor-targeting ligand construct. It is an amino acid sequence. The sequence is: SANSNPAMAPRERKAGCKNFFWKTFTSCGGGGSGGGGSGGGGSSANSNPAMAPRERKAGCKNFFWKTFTSC.

[0395] SEQ ID NO: 59 includes target ligands: 1-71.

[0396] SEQ ID NO: 60 is the sequence name of the 2xSST28 3xg4S ligand. It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCGAACAGCAACCCGGCGATGGCGCCGCGCGAACGCAAAGCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCGGCGGCGGCGGCAGCGGCGGCGCGGCAGCGGCGGCGGCGGCAGCAG CGCGAACAGCAACCCGGCGATGGCGCCGCGCGAACGCAAAGCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCGGCGGCGGCGGCAGCGGCGGCGCGGCAGCGGCGGCGGCGGCAGC.

[0397] SEQ ID NO: 60 includes target ligand: 1-213; free energy: -117; gdT CAI: 0.603084331934136; number of ORFs: 0.

[0398] SEQ ID NO: 61 is the sequence name of the 2xSST28 3xg4S ligand ECOg(192). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCTGCCAACTCCAACCCCGCTATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGCTCAGGAGGCGGAGGCTCCGGAGGAGGCGGCTCCTC CGCCAACTCCAACCCCGCTATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGCTCCGGAGGAGGCGGCTCAGGAGGCGGCGGCAGC.

[0399] SEQ ID NO: 61 includes target ligand: 1-213; free energy: -138.2; gdT CAI: 0.800017929945784; number of ORFs: 0.

[0400] SEQ ID NO: 62 is the sequence name of the 2xSST28 3xg4S ligand ECOg(141). This is a tumor-targeting ligand construct. This is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCCGCTATGGCTCCCGGGACGGAAGGCTGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGTTCCGGAGGCGGAGGTTCCGGCGGAGGCGGCTCCTC CGCCAACTCCAACCCCGCGATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGTTCCGGCGGAGGAGGTTCCGGAGGCGGCGGCTCC.

[0401] SEQ ID NO: 62 includes target ligand: 1-213; free energy: -146.7; gdT CAI: 0.786043171489395; number of ORFs: 0.

[0402] SEQ ID NO: 63 is the sequence name of the 2xSST28 3xg4S ligand ECOg(241). This is a tumor-targeting ligand construct. This is a DNA sequence. The sequence is: AGCGCCAACTCCAACCCCGCTATGGCTCCCGGGACGCAAGGCTGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGCTCTGGAGGCGGAGGCTCTGGCGGAGGCGGCTCCTC CGCCAACTCCAACCCCGCGATGGCTCCCAGGGAGCGCAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCGGAGGCTCTGGCGGAGGAGGCTCTGGAGGCGGCGGCTCC.

[0403] SEQ ID NO: 63 includes target ligand: 1-213; free energy: -142.6; gdT CAI: 0.788525759670669; number of ORFs: 0.

[0404] SEQ ID NO: 64 is the sequence name of the 2xSST28 3xg4S ligand ECOg(172). This is a tumor-targeting ligand construct. This is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCGGCCATGGCTCCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGCTCCTC GGCCAACTCCAACCCGGCCATGGCTCCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGATCT.

[0405] SEQ ID NO: 64 includes target ligand: 1-213; free energy: -140; gdT CAI: 0.719392416533176; number of ORFs: 0.

[0406] SEQ ID NO: 65 is the sequence name of the 2xSST28 3xg4S ligand ECOg(266). This is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCCGCTATGGCTCCCGGGACGGAAGGCTGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGTGGCGGCGGCGGATCTGGCGGAGGAGGCTCTGGCGGAGGCGGCTCCTC CGCCAACTCCAACCCCGCTATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGTGGCGGCGGCGGATCTGGCGGAGGAGGCTCTGGCGGAGGCGGAAGC.

[0407] SEQ ID NO: 65 includes target ligand: 1-213; free energy: -135.3; gdT CAI: 0.799023029714678; number of ORFs: 0.

[0408] SEQ ID NO: 66 is the sequence name of the 2xSST28 2xg4S ligand. It is a tumor-targeting ligand construct. It is an amino acid sequence. The sequence is: SANSNPAMAPRERKAGCKNFFWKTFTSCGGSGGSGGSGGSANSNPAMAPRERKAGCKNFFWKTFTSCGGSGGSGGSGG.

[0409] SEQ ID NO: 66 includes target ligand: 1-70; central linker: 71-84.

[0410] SEQ ID NO: 67 is the sequence name of the 2xSST28 2xg4S ligand. It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCGAACAGCAACCCGGCGATGGCGCCGCGCGAACGCAAAGCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAG CGCGAACAGCAACCCGGCGATGGCGCCGCGCGAACGCAAAGCGGGCTGCAAAAACTTTTTTTGGAAAACCTTTACCAGCTGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGC.

[0411] SEQ ID NO: 67 includes: target ligand: 1-210; central linker: 221-252; free energy: -110.4; gdT CAI: 0.602525074392084; number of ORFs: 0.

[0412] SEQ ID NO: 68 is the sequence name of the 2xSST28 2xg4S ligand ECOg(114). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCCGCAATGGCTCCCGGGGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCAGCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTC CGCCAACTCCAACCCCGCAATGGCTCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCAGCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGC.

[0413] SEQ ID NO: 68 includes target ligand: 1-210; central linker: 221-252; free energy: -140.6; gdT CAI: 0.815587600211903; number of ORFs: 0.

[0414] SEQ ID NO: 69 is the sequence name of the 2xSST28 2xg4S ligand ECOg(86). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCTGCCAATTCCAACCCCGCGATGGCCCCTCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTC TGCCAATTCCAACCCCGCGATGGCCCCTCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGC.

[0415] SEQ ID NO: 69 includes target ligand: 1-210; central linker: 221-252; free energy: -146.5; gdT CAI: 0.793448781755408; number of ORFs: 0.

[0416] SEQ ID NO: 70 is the sequence name of the 2xSST28 2xg4S ligand ECOg(132). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCCGCGAACTCCAACCCCGCGATGGCTCCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACGTTCACGTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTC CGCGAACTCCAACCCCGCGATGGCTCCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACGTTCACGTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGA.

[0417] SEQ ID NO: 70 includes target ligand: 1-210; central linker: 221-252; free energy: -141.6; gdT CAI: 0.768520163043281; number of ORFs: 0.

[0418] SEQ ID NO: 71 is the sequence name of the 2xSST28 2xg4S ligand ECOg(131). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCCAACTCCAACCCCGCGATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTC CGCCAACTCCAACCCCGCGATGGCTCCCAGGGAGCGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGTGGC.

[0419] SEQ ID NO: 71 includes target ligand: 1-210; central linker: 221-252; free energy: -140.8; gdT CAI: 0.805786107124917; number of ORFs: 0.

[0420] SEQ ID NO: 72 is the sequence name of the 2xSST28 2xg4S ligand ECOg(137). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCCGCTATGGCCCCTAGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTC CGCCAACTCCAACCCCGCTATGGCCCCTAGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGA.

[0421] SEQ ID NO: 72 includes target ligand: 1-210; central linker: 221-252; free energy: -140; gdT CAI: 0.822267579371957; number of ORFs: 1.

[0422] SEQ ID NO: 73 is the sequence name of the SST28 ligand. It is a tumor-targeted ligand construct. It is an amino acid sequence. The sequence is: SANSNPAMAPRERKAGCKNFFWKTFTSC.

[0423] SEQ ID NO: 73 includes target ligands: 1-28.

[0424] SEQ ID NO: 74 is the sequence name of the SST28 ligand. It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: TCCGCCAACTCCAACCCGGCCATGGCTCCCCGGGAGAGGAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCAGCTGC.

[0425] SEQ ID NO: 74 includes target ligands: 1-84; free energy: -26.1; gdT CAI: 0.909139392619506; number of ORFs: 0.

[0426] SEQ ID NO: 75 is the sequence name of the SST28 ligand ECOg(10). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCCAACAGCAACCCCGCTATGGCTCCCAGGGAGCGCAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCTTGC.

[0427] SEQ ID NO: 75 includes target ligands: 1-84; free energy: -27.4; gdT CAI: 0.925222356313033; number of ORFs: 0.

[0428] SEQ ID NO: 76 is the sequence name of the SST28 ligand ECOg(172). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCGAACAGCAACCCGGCCATGGCCCCTCGCGAGCGAAAGGCCGGGTGCAAGAACTTCTTCTGGAAGACCTTCACCTCGTGC.

[0429] SEQ ID NO: 76 includes target ligands: 1-84; free energy: -31.1; gdT CAI 0.762381535851502; number of ORFs: 0.

[0430] SEQ ID NO: 77 is the sequence name of the SST28 ligand ECOg(38). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCCAACTCCAACCCGGCTATGGCGCCCAGGGAGAGGAAGGCCGGCTGCAAGAACTTCTTCTGGAAGACCTTCACCTCCTGC.

[0431] SEQ ID NO: 77 includes target ligands: 1-84; free energy: -31; gdT CAI: 0.866223933524215; number of ORFs: 0.

[0432] SEQ ID NO: 78 is the sequence name of the SST28 ligand ECOg(5). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGTGCGAACTCGAACCCGGCCATGGCGCCCAGAGAGCGCAAGGCCGGGTGCAAGAACTTTTTCTGGAAAACGTTCACATCGTGC.

[0433] SEQ ID NO: 78 includes target ligands: 1-84; free energy: -30.7; gdT CAI: 0.714442287269154; number of ORFs: 0.

[0434] SEQ ID NO: 79 is the sequence name of the SST28 ligand ECOg(44). It is a tumor-targeting ligand construct. It is a DNA sequence. The sequence is: AGCGCGAACTCGAACCCGGCCATGGCTCCGCGCGAGCGTAAGGCCGGGTGCAAGAACTTTTTCTGGAAAACCTTCACGAGTTGT.

[0435] SEQ ID NO: 79 includes target ligands: 1-84; free energy: -30.1; gdT CAI: 0.688444421590185; number of ORFs: 0.

[0436] SEQ ID NO: 80 is the sequence name of TPO. It is a tumor targeting ligand construct. It is an amino acid sequence. The sequence is: SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPSRTSLVLTLNELG.

[0437] SEQ ID NO: 80 includes target ligands: 1-171.

[0438] SEQ ID NO: 81 is the sequence name of TPO. It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCCCCAGCTCCACCAGCCTGTGACCTGAGGGTGCTGAGCAAGCTCCTGAGGGACTCCCATGTGCTGCACAGCAGGCTGAGCCAGTGCCCTGAGGTGCATCCCCTGCCAACCCCTGTGCTGCTGCCAGCT GTGGACTTCTCCCTTGGGGAGTGGAAGACCCAGATGGAGGAGACCAAGGCCCAGGACATCCTTGGGGCTGTGACCCTGCTGCTGGAAGGGGTGATGGCTGCCAGGGGCCAGCTGGGGCCAACCTGCCTC AGCTCCCTGCTGGGGCAGCTGTCAGGGCAGGTGAGGCTGCTGCTGGGAGCCCTGCAGTCCCTGCTGGGGACCCAGGGCAGGACCACAGCCCACAAGGACCCCAATGCCATCTTCCTGAGCTTCCAGCAC CTGCTGAGGGGCAAGGTGAGGTTCCTGATGCTGGTTGGAGGCAGCACCCTGTGTGTCAGGAGAGCTCCACCAACCACAGCTGTGCCCAGCAGGACCAGCCTGGTGCTGACCCTGAATGAGCTTGGA.

[0439] SEQ ID NO: 81 includes target ligand: 1-513; free energy: -251.6; gdT CAI: 0.86805332586369; number of ORFs: 4.

[0440] SEQ ID NO: 82 is the sequence name of TPO ECOg(6). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: AGCCCCGCACCACCTGCCTGCGACCTGCGGGTGCTGTCCAAGCTGCTGCGGGACAGCCACGTGCTGCACAGCAGGCTGTCCCAGTGCCCCGAGGTGCACCCACTGCCCACGCCCGTGCTGCTGCCCGCT GTGGACTTCTCCCTGGGCGAGTGGAAGACACAGATGGAGGAGACCAAGGCCCAGGACATCCTGGGCGCCGTGACCCTGCTGCTGGAAGGGGTGATGGCCGCCAGAGGGCAGCTGGGGCCAACGTGCCTG TCCTCACTGCTGGGGCAGCTGTCCGGGCAGGTGCGGCTGCTGCTGGGCGCCCTGCAGTCCCTGCTGGGCACCCAGGGGCGCACCACAGCTCACAAGGACCCCAACGCCATCTTCCTGTCCTTCCAGCAC CTGCTGCGGGGCAAGGTGCGGTTCCTGATGCTGGTCGGCGGCAGCACCCTGTGCGTGCGCAGGGCACCACCGACCACAGCTGTGCCCAGCAGGACCTCACTGGTGCTGACCCTGAACGAGCTGGGC.

[0441] SEQ ID NO: 82 includes target ligand: 1-513; free energy: -258.8; gdT CAI: 0.891232089689473; number of ORFs: 1.

[0442] SEQ ID NO: 83 is the sequence name of TPO ECOg(42). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: AGCCCGGCTCCTCCGGCCTGCGACCTGCGCGTGCTGAGCAAGCTCCTGCGGGACTCGCACGTGCTGCACTCGCGCCTGAGCCAGTGCCCCGAGGTGCATCCCCTGCCTACCCCGGTGCTCCTGCCCGCG GTGGACTTCTCGCTCGGGGAGTGGAAGACCCAGATGGAGGAGACCAAGGCCCAGGACATACTCGGGGCCGTGACCCTGCTCCTGGAAGGGGTCATGGCAGCTCGGGGCCAGCTCGGGCCTACGTGCCTG AGCTCCCTGCTCGGGCAGCTGTCCGGGCAGGTCCGGCTCCTGCTCGGGGCCCTGCAGAGCCTGCTCGGGACCCAGGGCCGACCACGGCTCACAAGGACCCGAACGCGATCTTCCTGAGCTTCCAGCAC CTGCTCGGGGCAAGGTCAGGTTCCTGATGCTGGTCGGAGGCTCGACCCTGTGCGTGCGCAGGGCTCCTCCGACCACGGCCGTGCCCTCGCGCACGAGCCTGGTCCTGACCCTGAACGAGCTCGGG.

[0443] SEQ ID NO: 83 includes target ligand: 1-513; free energy: -273.6; gdT CAI: 0.779630301042555; number of ORFs: 0.

[0444] SEQ ID NO: 84 is the sequence name of TPO ECOg(19). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: TCTCCGGCTCCGCCTGCCTGCGACCTGCGGGTGCTGTCGAAGCTGCTGCGGGACAGCCACGTCCTCCACAGCCGCCTGAGCCAGTGCCCGGAGGTGCACCCGCTGCCTACGCCGGTGCTGCTGCCGGCC GTGGACTTCAGCCTCGGGGAGTGGAAGACGCAGATGGAGGAGACCAAGGCCCAGGACATCCTCGGGGCCGTGACCCTGCTCCTGGAAGGGGTGATGGCAGCGCGAGGGCAGCTGGGGCCTACCTGCCTC AGCTCCCTGCTGGGGCAGCTGTCGGGGCAGGTGCGGCTGCTGCTCGGGGCCCTGCAGTCCCTGCTCGGGACCCAGGGCCGGACCACAGCCCACAAGGACCCCAACGCCATCTTCCTCTCCTTCCAGCAC CTGCTCGGGGCAAGGTCCGGTTCCTGATGCTGGTCGGCGGCAGCACCCTGTGCGTGAGACGGGCTCCGCCTACCACGGCCGTGCCCTCGCGCACGAGCCTGGTCCTGACCCTGAACGAGCTCGGG.

[0445] SEQ ID NO: 84 includes target ligand: 1-513; free energy: -269.4; gdT CAI: 0.79850475511585; number of ORFs: 1.

[0446] SEQ ID NO: 85 is the sequence name of TPO ECOg(53). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: AGCCCCGCGCCGCCAGCGTGCGATCTGCGCGTGCTGAGCAAGCTGCTGCGCGACTCGCACGTGCTGCACTCGCGGCTCTCGCAGTGCCCCGAGGTGCACCCGCTGCCCACACCCGTGCTGCTGCCCGCG GTGGACTTCTCGCTCGGCGAGTGGAAGACGCAGATGGAGGAGACGAAAGCGCAGGACATCCTCGGCGCGGTGACGCTGCTGCTCGAAGGCGTGATGGCTGCTCGGGCAGCTCGGGCCTACGTGCCTG AGCTCGCTGCTCGGGCAGCTGAGCGGGCAGGTGCGGCTGCTGCTCGGCGCGCTGCAGTCGCTGCTCGGCACGCAGGGGCGCACCACAGCGCACAAGGACCCGAACGCGATCTTCCTGAGCTTCCAGCAC CTGCTGCGCGGGAAGGTGCGCTTCCTGATGCTCGTCGGCGGCAGCACGCTGTGCGTGCGCAGAGCGCCTCCGACCACCGCGGTGCCCTCGCGCACCTCGCTCGTGCTCACGCTGAACGAGCTCGGC.

[0447] SEQ ID NO: 85 includes target ligand: 1-513; free energy: -268.8; gdT CAI: 0.737843607302898; number of ORFs: 1.

[0448] SEQ ID NO: 86 is the sequence name of TPO ECOg(57). It is a tumor targeting ligand construct. It is a DNA sequence. The sequence is: AGCCCCGCTCCGCCAGCCTGCGACCTGCGGGTGCTGAGCAAGCTGCTGCGGGACAGCCACGTGCTGCACAGCCGGCTGAGCCAGTGCCCCGAGGTGCACCCGCTGCCCACGCCCGTGCTGCTGCCCGCT GTGGACTTCAGCCTGGGCGAGTGGAAGACCCAGATGGAGGAGACCAAGGCCCAGGACATCCTGGGCGCCGTGACCCTGCTGCTGGAAGGGGTGATGGCCGCTAGGGGCCAGCTGGGGGCCTACCTGCCTG AGCAGCCTGCTGGGGCAGCTGAGCGGGCAGGTGCGGCTGCTGCTGGGCGCCTGCAGAGCCTGCTGGGCACCCAGGGGCGCACCACAGCCCACAAGGACCCCAACGCCATCTTCCTGAGCTTCCAGCAC CTGCTGCGGGGCAAGGTGCGCTTCCTGATGCTGGTCGGCGGCAGCACCCTGTGCGTGCGCAGGGCTCCGCCTACCACCGCCGTGCCCAGCCGCACCAGCCTGGTGCTGACCCTGAACGAGCTCGGC.

[0449] SEQ ID NO: 86 includes target ligand: 1-513; free energy: -267.9; gdT CAI: 0.853835989559969; number of ORFs: 2.

[0450] SEQ ID NO: 87 is the sequence name of the Fc fusion site construct. It is an amino acid sequence. The sequence is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0451] SEQ ID NO: 87 includes fusion sites: 1-227.

[0452] SEQ ID NO: 88 is the sequence name of the Fc. It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0453] SEQ ID NO: 88 includes fusion sites: 1 to 681; free energy: -199.9; gdT CAI: 0.747707161534413; number of ORFs: 6.

[0454] SEQ ID NO: 89 is the sequence name of FC ECOg(85). It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0455] SEQ ID NO: 89 includes fusion site: 1-681; free energy: -269.2; gdT CAI: 0.894515513769793; number of ORFs: 0.

[0456] SEQ ID NO: 90 is the sequence name of FC ECOg(59). It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0457] SEQ ID NO: 90 includes fusion site: 1-681; free energy: -299.3; gdT CAI: 0.760430405503452; number of ORFs: 0.

[0458] SEQ ID NO: 91 is the sequence name of FC ECOg(5). It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0459] SEQ ID NO: 91 includes fusion sites: 1 to 681; free energy: -278.1; gdT CAI: 0.766513451450202; number of ORFs: 0.

[0460] SEQ ID NO: 92 is the sequence name of FC ECOg(23). It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0461] SEQ ID NO: 92 includes fusion site: 1-681; free energy: -276.3; gdT CAI: 0.769448727316174; number of ORFs: 0.

[0462] SEQ ID NO: 93 is the sequence name of FC ECOg(56). It is a fusion site construct. It is a DNA sequence. The sequence is: is.

[0463] SEQ ID NO: 93 includes fusion site: 1-681; free energy: -275.4; gdT CAI: 0.816569307205555; number of ORFs: 0.

[0464] SEQ ID NO: 94 is the sequence name of the Hum2 scFv. It is a gdT-targeting scFv construct. It is an amino acid sequence. The sequence is: IQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYNQKFKDRATLTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGSGGSGGSGGSGGDIQLTQSPSSLSASVGDRVTITCRASSSVSYMNWYQQKPGKAPKRWIYDTSKVASGAPSRFTGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPLTFGGGTKLEIK.

[0465] SEQ ID NO: 94 includes gdT scFv: 1 to 238; gdT VH: 119 to 132; gdT linker: 119 to 132; and gdT VL: 133 to 238.

[0466] SEQ ID NO: 95 is the sequence name of Hum2 scFv ECOg(0). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0467] SEQ ID NO: 95 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 397 to 714; free energy: -325.7; gdT CAI: 0.910798811448247; number of ORFs: 0.

[0468] SEQ ID NO: 96 is the sequence name of Hum2 scFv ECOg(183). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0469] SEQ ID NO: 96 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 97 to 714; free energy: -331.6; gdT CAI: 0.766742326650226; number of ORFs: 2.

[0470] SEQ ID NO: 97 is the sequence name of Hum2 scFv ECOg(6). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0471] SEQ ID NO: 97 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 397 to 714; free energy: -331; gdT CAI: 0.825626192459745; number of ORFs: 1.

[0472] SEQ ID NO: 98 is the sequence name of Hum2 scFv ECOg(42). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0473] SEQ ID NO: 98 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 397 to 714; free energy: -330; gdT CAI: 0.887375945709965; number of ORFs: 1.

[0474] SEQ ID NO: 99 is the sequence name of Hum2 scFv ECOg(196). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0475] SEQ ID NO: 99 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 397 to 714; free energy: -324.7; gdT CAI: 0.892144940351689; number of ORFs: 1.

[0476] SEQ ID NO: 100 is the sequence name of Hum2 scFv ECOg(172). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0477] SEQ ID NO: 100 includes gdT scFv: 1 to 714; gdT VH: 355 to 396; gdT linker: 355 to 396; gdT VL: 397 to 714; free energy: -324.5; gdT CAI: 0.8278596643083; number of ORFs: 0.

[0478] SEQ ID NO: 101 is the sequence name of gd-c V6 scFv. It is a gdT-targeting scFv construct. It is an amino acid sequence. The sequence is: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSKGLEWLGRTYYRSKWYNEYAASVKSRMSINPDTSKNQFSLQLNSVTPEDTALYYCARDLWELREACDIWGQGTMVTVSSGGSGGSGGSGGSGGDIVMTQSPSFLSTFVGDRVTITCRASQGISSYLAWYQQKPGKVPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPFTFGPGTKVDIK.

[0479] SEQ ID NO: 101 includes gdT scFv: 1 to 244; gdT VH: 124 to 137; gdT linker: 124 to 137; and gdT VL: 138 to 244.

[0480] SEQ ID NO: 102 is the sequence name of gd-c V6 scFv. It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0481] SEQ ID NO: 102 includes gdT scFv: 1 to 732; gdT VH: 369 to 411; gdT linker: 369 to 411; gdT VL: 412 to 732; free energy: −325.2; gdT CAI: 0.901042; and number of ORFs: 1.

[0482] SEQ ID NO: 103 is the sequence name of gd-c V6 scFv ECOg(69). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is CAGGTGCAGCTGCAGCAGTCCGGGCCCGGGCTGGTGAAGCCTTCCCAGACCCTCTCCCTCACCTGCGCCATCTCCGGGGATTCCGTGTCCTCCAACCGCGCCGCCTGGAACTGGATCAGGCAGTCCCCTTCCAAGGGGCTGGAGTGGCTGGGGCGCACGTACTACCGCTCCAAGTGGTACAACGAGTACGCCGCTTCCGTGAAGTCCCGCATGAGCATCAACCCCGATACCTCCAAGAACCAGTTCTCCCTGCAGCTGAACTCCGTGACCCCGGAGGATACCGCGCTGTACTACTGCGCCCGGGACCTGTGGGAGCTGCGGGAAGCCTGCGACATCTGGGGCCAGGGGACCATGGTGACCGTGTCCTCCGGAGGCTCCGGAGGCTCCGGAGGTTCCGGAGGCTCCGGCGGCGACATCGTGATGACGCAGTCCCCTTCCTTCCTGAGCACCTTCGTAGGGGACCGCGTTACCATCACCTGCAGGGCTTCCCAGGGGATCTCCTCCTACCTCGCATGGTACCAGCAGAAGCCCGGGAAGGTTCCCAAGCTGCTGATCTACGTGGCTTCCACGCTGCAGTCCGGGGTGCCTTCCCGGTTCTCCGGGAGCGGGAGCGGGACGGAGTTCACCCTCACCATCAGCTCCCTGCAGCCCGAGGACTTCGCAACGTACTACTGCCAGCAGCTGAACTCCTACCCCTTCACCTTCGGGCCCGGGACGAAGGTGGACATCAAG.

[0483] SEQ ID NO: 103 includes gdT scFv: 1 - 732; gdT VH: 369 - 411; gdT linker: 369 - 411; gdT VL: 412 - 732; free energy: -328.2; gdT CAI: 0.885003557188278; number of ORFs: 0.

[0484] SEQ ID NO: 104 is the sequence name of gd-c V6 scFv ECOg(34). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0485] SEQ ID NO: 104 includes gdT scFv: 1 to 732; gdT VH: 369 to 411; gdT linker: 369 to 411; gdT VL: 412 to 732; free energy: -353.6; gdT CAI: 0.744822337548797; number of ORFs: 2.

[0486] Array number 105 is the sequence name of gd-c V6 scFv ECOg(55). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: CAGGTGCAGCTGCAGCAGAGCGGCCCCGGGCTCGTGAAGCCGTCGCAGACCCTGAGCCTCACCTGCGCCATCTCCGGGGACTCCGTGTCCTCGAACCGCGCCGCGTGGAACTGGATTCGGCAGAGCCCCAGCAAGGGCCTGGAGTGGCTGGGGCGCACCTACTACCGCTCCAAGTGGTACAACGAGTACGCCGCCTCGGTGAAGTCCCGGATGAGCATCAACCCCGACACCTCGAAGAACCAGTTCTCGCTGCAGCTGAACTCCGTGACCCCGGAGGACACGGCGCTGTACTACTGCGCCCGGGACCTCTGGGAGCTCCGCGAGGCCTGCGACATCTGGGGCCAGGGGACCATGGTCACCGTGTCCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCGACATCGTGATGACCCAGAGCCCCAGCTTCCTGAGCACCTTCGTCGGGGACCGGGTCACCATCACGTGCCGCGCGTCCCAGGGGATCTCCTCGTACCTGGCCTGGTACCAGCAGAAGCCCGGGAAGGTGCCGAAGCTGCTGATCTACGTGGCCTCGACGCTGCAGTCCGGGGTCCCGAGCCGCTTCAGCGGCTCCGGGTCCGGGACCGAGTTCACCCTGACCATCTCGTCGCTGCAGCCCGAGGACTTCGCCACGTACTACTGCCAGCAGCTGAACAGCTACCCCTTCACCTTCGGCCCCGGGACCAAGGTGGACATCAAG.

[0487] Accession number 105 includes gdT scFv: 1 - 732; gdT VH: 369 - 411; gdT linker: 369 - 411; gdT VL: 412 - 732; free energy: -351.8; gdT CAI: 0.812038056353435; number of ORFs: 0.

[0488] Accession number 106 is the sequence name of gd-c V6 scFv ECOg(21). It is a gdT target scFv construct. It is a DNA sequence. The sequence is: CAGGTGCAGCTGCAGCAGAGCGGGCCCGGGCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCGCCATCTCCGGGGACAGCGTGAGCAGCAACCGGGCCGCCTGGAACTGGATCAGGCAGAGCCCCAGCAAGGGGCTGGAGTGGCTGGGGCGCACATACTACCGCTCCAAGTGGTACAATGAGTATGCCGCCAGCGTGAAGAGCCGCATGAGCATCAACCCCGACACCTCCAAGAACCAGTTCAGCCTGCAGCTGAACAGCGTGACGCCCGAGGACACTGCCCTGTACTACTGCGCCAGGGACCTGTGGGAGCTGCGCGAGGCCTGCGACATCTGGGGCCAGGGCACCATGGTGACAGTGTCCTCCGGCGGCTCAGGAGGCTCCGGAGGCTCTGGCGGCTCAGGCGGCGACATCGTGATGACCCAGAGCCCCAGCTTCCTGAGCACCTTCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGTGCCCAAGCTGCTGATCTATGTGGCCAGCACCCTGCAGAGCGGGGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACAGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACATACTACTGCCAGCAGCTGAACAGCTACCCCTTCACCTTCGGGCCCGGCACCAAGGTGGACATCAAG.

[0489] SEQ ID NO: 106 includes gdT scFv: 1 to 732; gdT VH: 369 to 411; gdT linker: 369 to 411; gdT VL: 412 to 732; free energy: −338.7; gdT CAI: 0.860184134678756; number of ORFs: 3.

[0490] SEQ ID NO: 107 is the sequence name of gd-c V6 scFv ECOg(99). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: CAGGTGCAGCTGCAGCAGAGCGGGCCCGGGCTCGTGAAGCCCTCGCAGACCCTCTCCCTCACGTGCGCGATCTCCGGGGACTCCGTGTCCTCCAACCGCGCCGCGTGGAACTGGATACGGCAGAGCCCCTCGAAGGGGCTGGAGTGGCTGGGGCGCACGTACTACCGCTCCAAGTGGTACAACGAGTACGCCGCCTCCGTGAAGTCCCGCATGAGCATCAACCCCGACACCTCCAAGAACCAGTTCTCCCTGCAGCTGAACTCCGTGACTCCCGAGGACACCGCGCTGTACTACTGCGCGCGGGACCTGTGGGAGCTGCGCGAGGCGTGCGACATCTGGGGACAGGGGACCATGGTGACCGTGTCCTCCGGAGGAAGCGGAGGAAGCGGAGGAAGCGGAGGAAGCGGAGGAGACATCGTGATGACGCAGTCCCCTTCCTTCCTCTCCACCTTCGTGGGAGACCGCGTGACCATCACGTGCCGCGCTTCCCAGGGGATCTCCTCCTACCTCGCGTGGTACCAGCAGAAGCCCGGGAAGGTGCCCAAGCTCCTCATCTACGTGGCCTCCACGCTGCAGAGCGGGGTGCCCTCGCGCTTCTCCGGGAGCGGGAGCGGGACGGAGTTCACCCTCACCATCTCTTCCCTGCAGCCCGAGGACTTCGCCACGTACTACTGCCAGCAGCTGAACTCCTACCCCTTCACCTTCGGGCCCGGGACGAAGGTGGACATCAAG.

[0491] SEQ ID NO: 107 includes gdT scFv: 1-732; gdT VH: 369-411; gdT linker: 369-411; gdT VL: 412-732; free energy: -335; gdT CAI: 0.835639819762746; number of ORFs: 0.

[0492] SEQ ID NO: 108 is the sequence name of gd-c V1 HL scFv. It is a gdT-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLLESGGGLVKPGGSLRLSCAASRFTLSSYDMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGVGGTDYYYYGLDVWGQGTTVTVSSGGSGGSGGSGGEIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK.

[0493] SEQ ID NO: 108 includes gdT scFv: 1 to 248; gdT VH: 126 to 139; gdT linker: 126 to 139; and gdT VL: 140 to 248.

[0494] SEQ ID NO: 109 is the sequence name of gd-c V1 HL scFv. It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGAAACCCGGCGGCTCCCTGCGGCTGTCCTGCGCCGCCTCCAGGTTCACCCTGTCCAGCTACGACATGAACTGGGTGAGGCAGGCTCCCGGGAAGGGGCTGGAGTGGGTGTCCTCCATCTCCTCCAGCTCCAGCTACATCTACTACGCCGATTCCGTGAAGGGGAGATTCACCATCTCCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACAGGGGCGTTGGCGGCACCGACTACTACTACTACGGGCTGGACGTGTGGGGCCAGGGGACCACCGTGACCGTGTCCAGCGGCGGCTCTGGCGGCTCTGGAGGCTCTGGCGGCTCTGGCGGCGAGATCGTGATGACCCAGTCTCCCGGGACCCTGTCCCTGTCTCCCGGGGAGAGGGCTACCCTGTCCTGCAGGGCCAGCCAGTCCGTGTCCTCCAGCTACCTGGCCTGGTACCAGCAGAAACCCGGGCAGGCTCCCCGGCTGCTGATCTACGGGGCCTCTTCCAGGGCCACCGGCATCCCCGACAGGTTCTCCGGGTCCGGGTCCGGGACCGACTTCACCCTGACCATCTCCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGGTCCTCTCCACCTTACACCTTCGGGCAGGGGACCAAGGTGGAGATCAAG.

[0495] SEQ ID NO: 109 includes gdT scFv: 1 - 744; gdT VH: 376 - 417; gdT linker: 376 - 417; gdT VL: 418 - 744; free energy: -341.2; gdT CAI: 0.91246469035254; number of ORFs: 2.

[0496] Array number 110 is the sequence name of gd-c V1 HL scFv(63). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: GAGGTCCAGCTCCTGGAGAGCGGAGGAGGCCTGGTGAAGCCCGGAGGCTCCCTGAGGCTCTCCTGCGCCGCCTCCAGGTTCACCCTCTCCTCCTACGACATGAACTGGGTGAGGCAGGCCCCGGGGAAGGGGCTGGAGTGGGTCTCCTCCATCTCCTCCTCCTCCTCCTACATCTACTACGCCGACTCCGTGAAGGGGCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTCTACCTCCAGATGAACTCCCTGAGGGCCGAGGACACGGCCGTCTACTACTGCGCCCGGGACCGAGGGGTAGGAGGCACCGACTACTACTACGGGCTGGACGTCTGGGGCCAGGGGACCACCGTGACCGTCTCCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGCTCCGGAGGAGAGATCGTGATGACCCAGAGCCCCGGGACCCTCTCCCTGAGCCCCGGGGAGAGGGCTACCCTCTCCTGCCGGGCCAGCCAGAGCGTCTCCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCGGGGCAGGCCCCTAGGCTCCTGATCTACGGGGCCTCCTCTAGGGCCACCGGCATCCCGGACCGCTTCTCCGGGTCCGGGTCCGGGACCGACTTCACCCTCACCATCTCCCGGCTGGAGCCGGAGGACTTCGCCGTCTACTACTGCCAGCAGTACGGGTCCTCTCCTCCTTACACCTTCGGGCAGGGGACCAAGGTGGAGATCAAG.

[0497] Array number 110 includes gdT scFv: 1~744; gdT VH: 376~417; gdT linker: 376~417; gdT VL: 418~744; free energy: -362.6; gdT CAI: 0.854971378798751; number of ORFs: 1.

[0498] Array number 111 is the sequence name of gd-c V1 HL scFv(72). It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: GAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGAAACCCGGCGGCAGCCTGCGGCTGTCCTGCGCGGCAAGCCGCTTCACGCTGTCCAGCTACGACATGAACTGGGTGCGCCAGGCACCCGGCAAGGGGCTGGAGTGGGTGTCCAGCATATCCAGCTCGTCAAGCTACATATACTACGCGGACAGCGTGAAGGGCCGGTTTACCATCTCGCGGGATAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCGGTGTACTACTGCGCAAGGGACCGCGGGGTAGGCGGCACGGATTACTACTACTACGGGCTGGACGTGTGGGGCCAGGGGACCACCGTGACGGTGTCCTCCGGCGGCTCAGGCGGTTCCGGTGGCTCTGGCGGCTCAGGCGGCGAGATTGTCATGACGCAGTCACCCGGCACGCTTAGCCTGTCGCCCGGGGAACGCGCCACGCTGTCCTGCCGGGCCAGCCAGTCGGTGTCCAGCAGCTACCTGGCGTGGTACCAGCAGAAACCCGGCCAGGCGCCCCGGCTGCTTATCTACGGGGCGTCTAGCCGGGCAACCGGCATCCCGGACCGCTTCAGCGGGTCGGGCAGCGGGACGGACTTCACGCTGACAATCAGCCGGCTGGAGCCCGAGGACTTCGCGGTGTACTACTGCCAGCAGTACGGCAGCTCGCCGCCTTACACGTTTGGCCAGGGCACCAAGGTGGAAATCAAG

[0499] SEQ ID NO: 111 includes gdT scFv: 1 to 744; gdT VH: 376 to 417; gdT linker: 376 to 417; gdT VL: 418 to 744; free energy: -366.6; gdT CAI: 0.761156065773582; number of ORFs: 4.

[0500] SEQ ID NO: 112 is the sequence name of gd-c V1 HL scFv (14). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0501] SEQ ID NO: 112 includes gdT scFv: 1 to 744; gdT VH: 376 to 417; gdT linker: 376 to 417; gdT VL: 418 to 744; free energy: -366.1; gdT CAI: 0.774531811261147; number of ORFs: 3.

[0502] SEQ ID NO: 113 is the sequence name of gd-c V1 HL scFv (22). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: GAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGAAACCCGGCGGCTCCCTGCGGCTGTCCTGCGCCGCCAGCAGGTTCACCCTCTCCTCCTACGACATGAACTGGGTGCGGCAGGCCCCAGGGAAGGGCCTGGAGTGGGTGAGCAGCATCAGCAGCAGCAGCAGCTACATCTACTACGCCGACAGCGTCAAGGGGCGCTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTCCGGGCCGAGGACACGGCCGTGTACTACTGCGCCCGTGATCGTGGCGTCGGCGGCACCGACTACTACTACTACGGGCTGGACGTGTGGGGCCAGGGGACGACCGTGACCGTCTCCTCCGGCGGCTCCGGAGGCTCTGGTGGTTCTGGCGGCTCCGGCGGCGAGATCGTCATGACCCAGAGCCCCGGGACCCTGTCCCTGAGCCCCGGGGAGAGGGCCACGCTCTCCTGCCGGGCCAGCCAGTCCGTGTCCTCCTCCTACCTGGCCTGGTACCAGCAGAAACCCGGCCAGGCGCCCAGGCTGCTGATCTACGGGGCCTCTTCTCGCGCCACGGGCATCCCCGACCGCTTCTCCGGGAGCGGCTCCGGGACGGACTTCACCCTGACCATCAGCCGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGGTCCTCGCCGCCGTACACCTTCGGGCAGGGGACGAAGGTGGAGATCAAG is as follows.

[0503] SEQ ID NO: 113 includes gdT scFv: 1 - 744; gdT VH: 376 - 417; gdT linker: 376 - 417; gdT VL: 418 - 744; free energy: -359.3; gdT CAI: 0.831881016856322; number of ORFs: 3.

[0504] SEQ ID NO: 114 is the sequence name of gd-c V1 HL scFv (11). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0505] SEQ ID NO: 114 includes gdT scFv: 1 to 744; gdT VH: 376 to 417; gdT linker: 376 to 417; gdT VL: 418 to 744; free energy: -358.3; gdT CAI: 0.705025041042151; number of ORFs: 1.

[0506] SEQ ID NO: 115 is the sequence name of the JAML scFv. It is a gdT-targeting scFv construct. It is an amino acid sequence. The sequence is: DVQLVESGAELVRPGASKLSCKALAYTFTDYEMHWVKQTPVHGLEWIGIIHPGSGGTVYNQKFKGKATLTADKSSSTAYMELSSLTSEDSTVYYCTRRRYYGSSYNWYFDVWGAGNGGSGGSGGSGGSGGVLTQSPASLAASVGETVTITCRASENIYYSLAWYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSLKINSMQPEDTATYFCEQTYDVPLTFGAGTKLEL.

[0507] SEQ ID NO: 115 includes gdT scFv: 1 to 234; gdT VH: 117 to 130; gdT linker: 117 to 130; gdT VL: 131 to 234; free energy: -328.2; gdT CAI: 0.850906150801958; number of ORFs: 2.

[0508] SEQ ID NO: 116 is the sequence name of the JAML scFv. It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0509] SEQ ID NO: 116 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -302; gdT CAI: 0.868315362715317; number of ORFs: 10.

[0510] SEQ ID NO: 117 is the sequence name of JAML scFv(88). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0511] SEQ ID NO: 117 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -323.6; gdT CAI: 0.915740281407777; number of ORFs: 1.

[0512] SEQ ID NO: 118 is the sequence name of JAML scFv(84). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0513] SEQ ID NO: 118 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -328.2; gdT CAI: 0.850906150801958; number of ORFs: 2.

[0514] SEQ ID NO: 119 is the sequence name of JAML scFv(44). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0515] SEQ ID NO: 119 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -318.2; gdT CAI: 0.934766339108284; number of ORFs: 2.

[0516] SEQ ID NO: 120 is the sequence name of JAML scFv(23). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0517] SEQ ID NO: 120 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -314.5; gdT CAI: 0.828248130876822; number of ORFs: 3.

[0518] SEQ ID NO: 121 is the sequence name of JAML scFv(78). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0519] SEQ ID NO: 121 includes gdT scFv: 1 to 702; gdT VH: 349 to 390; gdT linker: 349 to 390; gdT VL: 391 to 702; free energy: -314.4; gdT CAI: 0.837369130789258; number of ORFs: 3.

[0520] SEQ ID NO: 122 is the sequence name of the CDXAR ligand. It is a gdT targeting ligand construct. It is an amino acid sequence. The sequence is: LSITTPEEMIEKAKGETAYLPCKFTLSPEDQGPLDIEWLISPADNQKVDQVIILYSGDKIYDDYYPDLKGRVHFTSNDLKSGDASINVTNLQLSDIGTYQCKVKKAPGVANKKIHLVVLVKPSGARCYVDGSEEIGSDFKIKCEPKEGSLPLQYEWQKLSDSQKMPTSWLAGKMCHLQRAVRPLPEATSAVIIHPWGPCLLPTWKDIPRLSITKYQVKTLNALLRVRLSHLLR.

[0521] SEQ ID NO: 122 encompasses gdT ligand: 1-233.

[0522] SEQ ID NO: 123 is the sequence name of the CDXAR ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0523] SEQ ID NO: 123 includes gdT ligand: 1-233; free energy: -284.1; gdT CAI: 0.86860152001121; number of ORFs: 7.

[0524] SEQ ID NO: 124 is the sequence name of CDXAR ligand (6). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0525] SEQ ID NO: 124 includes gdT ligand: 1-233; free energy: -291.4; gdT CAI: 0.912300179663014; number of ORFs: 1.

[0526] SEQ ID NO: 125 is the sequence name of CDXAR ligand (57). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0527] SEQ ID NO: 125 includes gdT ligand: 1-233; free energy: -290.9; gdT CAI: 0.776501808272779; number of ORFs: 4.

[0528] SEQ ID NO: 126 is the sequence name of CDXAR ligand (56). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0529] SEQ ID NO: 126 includes gdT ligand: 1-233; free energy: -287.5; gdT CAI: 0.835151871227941; number of ORFs: 2.

[0530] SEQ ID NO: 127 is the sequence name of CDXAR ligand (73). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0531] SEQ ID NO: 127 includes gdT ligand: 1-233; free energy: -287.4; gdT CAI: 0.810082913563651; number of ORFs: 0.

[0532] SEQ ID NO: 128 is the sequence name of CDXAR ligand (63). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: is.

[0533] SEQ ID NO: 128 includes gdT ligand: 1-233; free energy: -286; gdT CAI: 0.861258772692867; number of ORFs: 0.

[0534] SEQ ID NO: 129 is the sequence name of the CD5 scFv. It is a gdT-targeting scFv construct. It is an amino acid sequence. The sequence is: EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIK.

[0535] SEQ ID NO: 129 includes gdT scFv: 1 to 240; gdT VH: 119 to 133; gdT linker: 119 to 133; and gdT VL: 134 to 240.

[0536] SEQ ID NO: 130 is the sequence name of the CD5 scFv. It is a gdT-targeted scFv construct. It is a DNA sequence. The sequence is: is.

[0537] SEQ ID NO: 130 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: -346.2; gdT CAI: 0.943955363575702; and number of ORFs: 8.

[0538] SEQ ID NO: 131 is the sequence name of CD5 scFv(11). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0539] SEQ ID NO: 131 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: -345.9; gdT CAI: 0.885961949519618; number of ORFs: 1.

[0540] SEQ ID NO: 132 is the sequence name of CD5 scFv(9). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0541] SEQ ID NO: 132 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: −344.1; gdT CAI: 0.8842887077719; number of ORFs: 1.

[0542] SEQ ID NO: 133 is the sequence name of CD5 scFv (41). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0543] SEQ ID NO: 133 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: -342.6; gdT CAI: 0.78445426326926; number of ORFs: 1.

[0544] SEQ ID NO: 134 is the sequence name of CD5 scFv(21). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0545] SEQ ID NO: 134 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: −338.3; gdT CAI: 0.760359380692982; number of ORFs: 1.

[0546] SEQ ID NO: 135 is the sequence name of CD5 scFv(29). It is a gdT-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0547] SEQ ID NO: 135 includes gdT scFv: 1 to 720; gdT VH: 355 to 399; gdT linker: 355 to 399; gdT VL: 400 to 720; free energy: −334.2; gdT CAI: 0.774216148765874; number of ORFs: 1.

[0548] SEQ ID NO: 136 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is an amino acid sequence. The sequence is: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

[0549] SEQ ID NO: 136 encompasses gdT ligand: 1-333.

[0550] SEQ ID NO: 137 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCACCAACAAGCAGTAGCACCAAAAGACGCAGCTTCAGTTAGAGCACCTCCTACTCGACCTACAGATGATATTGAATGGTATTAATAACTACAAAAATCCTAAATTGACTCGAATGTTGACATTTAAATTTTATATGCCCAAAAAGGCAACCGAACTCAAGCATCTGCAGTGCCTGGAGGAGGAACTCAAGCCACTTGAA GAGGTCCTGAACCTGGCTCAGTCAAAAAATTTTCATCTGCGCCCCGGGACTTAATCAGCAATATCAACGTGATTGTTCTGGAGCTCAAGGGGTCTGAGACCACTTTTATGTGAATACGCTGATGAAACTGCGACAATCGTCGAGTTCCTCAATAGATGGATCACTTTCTGTCAATCCATTATTAGCACCCTGACC.

[0551] SEQ ID NO: 137 includes gdT ligand: 1-459; free energy: -92.6; gdT CAI: 0.7496927682812; number of ORFs: 2.

[0552] SEQ ID NO: 138 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCTCCTACCAGCTCCAGCACCAAGAAGACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATCCTGAACGGGATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTGCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAG GAGGTGCTGAACCTGGCCCAGAGCAAGAACTTCCACCTGCGCCCGGGACCTGATCAGCAACATCAACGTGATCGTGCTGGAGCTGAAGGGGTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTCCTGAACCGCTGGATCACCTTCTGCCAGAGCATCATCTCCACGCTGACC.

[0553] SEQ ID NO: 138 includes gdT ligand: 1-459; free energy: -140.2; gdT CAI: 0.948946971021626; number of ORFs: 0.

[0554] SEQ ID NO: 139 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCCCCGACCAGCAGCAGCACCAAGAAGACGCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATCCTGAACGGGATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCGACCGAGCTGAAGCACCTGCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAG GAGGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGCGGCCCGGACCTGATCAGCAACATCAACGTGATCGTGCTGGAGCTGAAGGGGTCCGAGACCACCTTCATGTGCGAGTACGCGGACGAGACCGCCACAATCGTGGAGTTCCTGAACCGCTGGATCACCTTCTGCCAGTCCATCATCAGCACCCTGACG.

[0555] SEQ ID NO: 139 includes gdT ligand: 1-459; free energy: -142.5; gdT CAI: 0.925399648745741; number of ORFs: 0.

[0556] SEQ ID NO: 140 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCCCCAACCTCCTCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATCCTGAACGGGATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTCACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTGCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAG GAGGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGCGGCCCGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAGCTGAAGGGGTCCGAGACCACCTTCATGTGCGAGTACGCAGATGAGACGGCTACAATCGTGGAGTTCCTGAACAGGTGGATCACCTTCTGCCAGTCCATCATCTCCACCTTGACA.

[0557] SEQ ID NO: 140 includes gdT ligand: 1-459; free energy: -139.3; gdT CAI: 0.966990185804835; number of ORFs: 1.

[0558] SEQ ID NO: 141 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCGCCCACGTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATCCTGAACGGGATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTGCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAG GAGGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGCGGCCCGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAGCTGAAGGGGTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTCCTGAACCGCTGGATCACCTTCTGCCAGAGCATCATCAGCACCCTCACC.

[0559] SEQ ID NO: 141 includes gdT ligand: 1-459; free energy: -136; gdT CAI: 0.957989396711122; number of ORFs: 0.

[0560] SEQ ID NO: 142 is the sequence name of the IL2r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: GCCCCTACCTCCTCTCCACCAAGAAGACCCAGCTCCAGCTGGAGCACCTCCTCTGGACCTCCAGATGATCCTCAACGGGATCAACAACTACAAGAACCCCAAGCTCACCCGGATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTCCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAG GAGGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTCCGGCCCAGGGACCTGATCTCCAACATCAACGTGATCGTCCTGGAGCTGAAGGGGTCCGAGACCACCTTCATGTGCGAGTACGCCGATGAGACAGCCACCATCGTGGAGTTCCTCAACAGGTGGATCACCTTCTGCCAGTCCATCATCAGCACCCTCACC.

[0561] SEQ ID NO: 142 includes gdT ligand: 1-459; free energy: -135.6; gdT CAI: 0.950256495713545; number of ORFs: 1.

[0562] SEQ ID NO: 143 is the sequence name of the IL15r ligand. It is a gdT targeting ligand construct. It is an amino acid sequence. The sequence is: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

[0563] SEQ ID NO: 143 encompasses gdT ligands: 1-114.

[0564] SEQ ID NO: 144 is the sequence name of the IL15r ligand. It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AACTGGGTTAACGTGATCAGCGATCTGAAGAAGATTGAAGATCTCATACAATCCATGCACATCGACGCTACCCTGTATACAGAGTCCGACGTTCACCCTAGCTGTAAGGTGACTGCCATGAAGTGCTTTTTACTGGAACTGCAGGTAATCAGTCTGGAGTCTGGTGATGCCTC AATTCACGACACGGTAGAGAATCTAATAATCCTTGCCAACAACTCTTTGAGTTCCAATGGCAATGTGACAGAATCTGGCTGCAAGGAGTGTGAAGAGCTTGAAGAGAAAAACATTAAAGAGTTCCTGCAATCCTTCGTGCATATAGTGCAGATGTTCATCAACACCTCG.

[0565] SEQ ID NO: 144 includes gdT ligand: 1-342; free energy: -86.8; gdT CAI: 0.773146219024413; number of ORFs: 3.

[0566] SEQ ID NO: 145 is the sequence name of the IL15r ligand (12). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AACTGGGTGAACGTGATCAGCGATCTGAAGAAGATCGAGGACCTGATCCAGTCCATGCACATCGACGCTACCCTGTACACCGAGTCGGACGTTCACCCCAGCTGCAAGGTGACCGCGATGAAGTGCTTCCTGCTCGAACTGCAGGTGATCAGCCTGGAGAGCGGGGACGCGAG CATCCACGATACGGTGGAGAACCTGATCATCCTGGCCAACAACTCGCTCAGCTCGAACGGGAACGTGACCGAGAGCGGGTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTCCAGTCGTTCGTGCACATCGTGCAGATGTTCATCAACACCTCC.

[0567] SEQ ID NO: 145 includes gdT ligand: 1-342; free energy: -125.8; gdT CAI: 0.877487412777548; number of ORFs: 0.

[0568] SEQ ID NO: 146 is the sequence name of the IL15r ligand (31). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AACTGGGTGAACGTCATCTCCGACCTGAAGAAGATCGAGGATCTGATCCAGTCGATGCACATCGACGCGACGCTCTACACCGAGTCGGACGTTCACCCCTCGTGCAAGGTCACGGCGATGAAGTGCTTCCTCCTGGAGCTGCAGGTGATCTCCCTGGAGTCGGGCGACGCCTC GATCCACGACACGGTCGAGAACCTGATCATCCTCGCGAACAACTCCCTCTCGTCCAACGGGAACGTGACCGAGAGCGGGTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTCCAGTCGTTCGTCCACATCGTCCAGATGTTCATCAACACCTCC.

[0569] SEQ ID NO: 146 includes gdT ligand: 1-342; free energy: -124.3; gdT CAI: 0.830363621275029; number of ORFs: 0.

[0570] SEQ ID NO: 147 is the sequence name of the IL15r ligand (29). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGTCCATGCACATCGACGCCACGCTGTACACCGAGTCCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCTCCCTGGAGTCCGGGGACGCCTC CATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACTCCCTGTCCTCCAACGGGAACGTGACCGAGTCCGGGTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACGTCG.

[0571] SEQ ID NO: 147 includes gdT ligand: 1-342; free energy: -123.2; gdT CAI: 0.953480352366457; number of ORFs: 0.

[0572] SEQ ID NO: 148 is the sequence name of the IL15r ligand (29). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AATTGGGTGAACGTCATCTCCGACCTCAAGAAGATCGAGGACCTCATCCAGTCCATGCACATCGACGCCACGCTCTACACGGAGTCCGACGTGCACCCGTCCTGCAAGGTGACGGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTCATCTCCTTGGAGTCCGGGGACGCCTC CATCCACGACACCGTCGAGAACCTCATCATCCTGGCCAACAACTCCTTGAGCTCCAACGGGAACGTGACGGAGTCCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCCTTCGTGCACATCGTGCAGATGTTCATCAACACGTCC.

[0573] SEQ ID NO: 148 includes gdT ligand: 1-342; free energy: -122.3; gdT CAI: 0.905077015244385; number of ORFs: 0.

[0574] SEQ ID NO: 149 is the sequence name of the IL15r ligand (32). It is a gdT targeting ligand construct. It is a DNA sequence. The sequence is: AACTGGGTGAACGTGATCTCCGACCTCAAGAAGATCGAGGACCTCATCCAGTCGATGCACATCGACGCGACCCTCTACACGGAGAGCGACGTCCATCCGAGCTGCAAGGTGACCGCGATGAAGTGCTTCCTCCTGGAGCTCCAGGTGATCTCCCTGGAGTCCGGGGACGCGAG CATCCACGACACCGTCGAGAACCTGATCATCCTCGCGAACAACTCGCTCTCCTCGAACGGGAACGTCACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTCGAAGAGAAGAACATCAAGGAGTTCCTCCAGAGCTTCGTCCACATCGTCCAGATGTTCATCAACACGTCG.

[0575] SEQ ID NO: 149 includes gdT ligand: 1-342; free energy: -122.1; gdT CAI: 0.814639976789479; number of ORFs: 0.

[0576] SEQ ID NO: 150 is the sequence name of B2m shRNA1. It is an HLA siRNA construct. It is a DNA sequence. The sequence is: It's GAATGGAGAGAGAATTGAA.

[0577] SEQ ID NO: 151 is the sequence name of CIITA shRNA7. It is an HLA siRNA construct. It is a DNA sequence. The sequence is: GCTCAGGCTAAGCTTGTACAA.

[0578] SEQ ID NO: 152 is the MND promoter. It is a DNA sequence. The sequence is: is.

[0579] SEQ ID NO: 153 is the HSPA8 promoter. It is a DNA sequence. The sequence is: CCCCTCCCTTCAGGCCCCGCGCGATTCCGCCCCCAGTTCTGTGCCGGCCAAGATCCCGGCTAGCGCCGCTATCATTGGTTAGTTCCAAGTTTGCCCGCCCCTCTTCCTCCTCCTTTTTCCGCCCCCTCCCTCCCGCGGAAGCTGGGGGCGCATGCGTAGAGGTGGACGCTCCCCTCCCCCGCCCGGGGTAACTGAGGACTCCCGCGCGCGGACTCGCTGCGCCCCACCCTCCCTTTCCCCGGGGCCGTCCGGAGAGCGGGGGCGAGCTTGAAAGTTCCAGAACGCTGCGGTGAGTGCGTTATCGTGAGGCGGAGCGCGGTGGGGTGGGTGCGGAAGGGGGCGAGGCCCGAGGAGTGGAGCCGGGCTTGTGATTGGGTCTTGTAAGGGCAGCCGGGCGTCTATTGGCCGGGGAAGCCGTAATGGCAGGCAGCAGGGGCGGGCCCCTTCTGGAAGGTTCTAAGATAGGGTATAAGAGGCAGGGTGGCGGGCGGAAACCGGTCTCATTGAACTCGCCTGCAGCTCTTGGGTTTTTTGTGGCTTCCTTCGTTATTGGAGCCAGGCCTACACCCCAGGTAAAACCTCTGCTCAAGAGTTGGGTTG is as follows.

[0580] SEQ ID NO: 154 is WPREmut, a DNA sequence. The sequence is: is.

[0581] SEQ ID NO: 155 is the amino acid sequence of 4GS. The sequence is: It's GGGGS.

[0582] SEQ ID NO: 156 is the sequence name of SSTR2-8. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLINSRNRKNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGMAWVRQAPGKGLEWVSFISNLGYSLYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTMVTVS.

[0583] SEQ ID NO: 156 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0584] SEQ ID NO: 157 is the sequence name of SSTR2-8 v1. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding the amino acid sequence of SEQ ID NO: 156. The sequence is: GAGATCGTGATGACCCAGAGCCCCGACTCCCTGGCCGTTTCCCTCGGGGAGCGGGCCACCATCTCCTGCAAGAGCTCCCAGAGCCTGATCAACTCCCGGAACCGGAAGAACTACCTGGCCTGGTACCAGCAGAACCCCGGGCAGCCGCCGAAGCTGCTGATCTACTGGGCCTCCACGCGGGAGAGCGGGGTGCCCGATCGCTTCTCCGGCTCCGGCTCCGGGACCGACTTCACCCTGACCATCTCCAGCCTGCAGGCCGAGGACGTGGCCGTCTACTACTGCAAGCAGAGCTACTACCTCTGGACCTTCGGGCAGGGGACCAAGGTGGAGATCAAAGGCGGCGGAGGCTCTGGCGGAGGAGGCTCTGGAGGCGGCGGCTCCCAGGTGCAGCTGGTGGAGAGCGGCGGAAGGCTGGTGCAGCCCGGCGGCTCCCTGAGGGTCTCCTGCGAGGCCTCCGGCTTCACCTTCTCCGACTACGGGATGGCCTGGGTGAGGCAGGCTCCCGGGAAGGGGCTGGAGTGGGTCTCCTTCATCTCCAACCTGGGCTACTCCCTGTACTACGCCGACTCCGTGAAGGGGCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTCTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTCTACTACTGCGCCCGGGCGCCCTACGACTACGACTCCTTCGACCCCATGGACTACTGGGGCCAGGGGACCATGGTGACCGTCTCC is as follows.

[0585] SEQ ID NO: 157 includes target scFv: 1-744; target VL: 1-336; target linker: 337-381; target VH: 382-744; free energy: -340.2; gdT CAI: 0.902; number of ORFs: 1.

[0586] SEQ ID NO: 158 is the sequence name of SSTR2-8 v2. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding the amino acid sequence of SEQ ID NO: 156. The sequence is: is.

[0587] SEQ ID NO: 158 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -322.5; gdT CAI: 0.915155531601317; number of ORFs: 0.

[0588] SEQ ID NO: 159 is the sequence name of SSTR2-8 v3. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 156. The sequence is: is.

[0589] SEQ ID NO: 159 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -320; gdT CAI: 0.913669760876946; number of ORFs: 0.

[0590] SEQ ID NO: 160 is the sequence name of SSTR2-8 v4. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 156. The sequence is: is.

[0591] SEQ ID NO: 160 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -319.399993896484; gdT CAI: 0.812998307048897; number of ORFs: 0.

[0592] SEQ ID NO: 161 is the sequence name of GD2-3. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: QVQLVQSGAEVEKPGASVKVSCKASGSSFTGYNMNWVRQAPGQGLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCVSGMKYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWYQQRPGQSPRLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPPLTFGQGTKLEIK.

[0593] SEQ ID NO: 161 includes target scFv: 1 to 248; target VL: 129 to 241; target linker: 114 to 128; and target VH: 1 to 113.

[0594] SEQ ID NO: 162 is the sequence name of GD2-3 v1. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 161. The sequence is: is.

[0595] SEQ ID NO: 162 includes target scFv: 1 to 723; target VL: 385 to 723; target linker: 340 to 384; target VH: 1 to 339; free energy: -335.6; gdT CAI: 0.914; and number of ORFs: 2.

[0596] SEQ ID NO: 163 is the sequence name of GD2-3 v2. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 161. The sequence is: CAGGTGCAGCTGGTGCAGTCCGGGGCCGAGGTGGAGAAGCCCGGCGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCTCCTCCTTCACCGGGTACAACATGAACTGGGTGAGACAGGCTCCCGGGCAGGGGCTGGAGTGGATCGGGGCCATCGACCCCTACTACGGCGGGACCAGCTACAACCAGAAGTTCAAGGGGAGGGCCACCCTGACAGTGGACAAGTCCATCTCCACCGCCTACATGGAGCTGTCCCGCCTGCGGTCCGACGACACCGCCGTGTACTACTGCGTGAGCGGGATGAAGTACTGGGGCCAGGGGACACTGGTGACCGTGTCCTCCGGCGGAGGCGGGTCCGGCGGAGGCGGGTCCGGCGGAGGCGGGTCCGACGTGGTGATGACCCAGTCTCCCCTGTCCCTGCCCGTGACCCTCGGCCAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCCTGGTGCACAGGAACGGGAACACATACCTGCACTGGTACCAGCAGCGGCCCGGCCAGTCTCCCCGCCTGCTGATCCACAAGGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGGTTCTCCGGCTCCGGCAGCGGGACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTCCCAGTCTACCCACGTGCCTCCCCTGACCTTCGGCCAGGGGACCAAGCTGGAGATCAAG.

[0597] SEQ ID NO: 163 includes target scFv: 1-723; target VL: 385-723; target linker: 340-384; target VH: 1-339; free energy: -346.600006103515; gdT CAI: 0.936000252358001; number of ORFs: 0.

[0598] SEQ ID NO: 164 is the sequence name of GD2-3 v3. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 161. The sequence is: is.

[0599] SEQ ID NO: 164 includes target scFv: 1 to 723; target VL: 385 to 723; target linker: 340 to 384; target VH: 1 to 339; free energy: -346.299987792968; gdT CAI: 0.950248810102486; number of ORFs: 0.

[0600] SEQ ID NO: 165 is the sequence name of GD2-3 v4. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 161. The sequence is: is.

[0601] SEQ ID NO: 165 includes target scFv: 1 to 723; target VL: 385 to 723; target linker: 340 to 384; target VH: 1 to 339; free energy: -340.600006103515; gdT CAI: 0.952480466768884; number of ORFs: 0.

[0602] SEQ ID NO: 166 is the sequence name of PTK7-14. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYLMYWIRQAPGKGLEWVSALGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPMYTFGPGTRVEIK.

[0603] SEQ ID NO: 166 includes target scFv: 1 to 236; target VL: 128 to 236; target linker: 113 to 127; and target VH: 1 to 112.

[0604] SEQ ID NO: 167 is the sequence name of PTK7-14 v1. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 166. The sequence is: is.

[0605] SEQ ID NO: 167 includes target scFv: 1-708; target VL: 382-708; target linker: 337-381; target VH: 1-336; free energy: -368.700012207031; gdT CAI: 0.910676800487004; number of ORFs: 2.

[0606] SEQ ID NO: 168 is the sequence name of PTK7-14 v2, a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 166. The sequence is: is.

[0607] SEQ ID NO: 168 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -367; gdT CAI: 0.932214679000254; number of ORFs: 0.

[0608] SEQ ID NO: 169 is the sequence name of PTK7-14 v3. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 166. The sequence is: is.

[0609] SEQ ID NO: 169 includes target scFv: 1-708; target VL: 382-708; target linker: 337-381; target VH: 1-336; free energy: -354.5; gdT CAI: 0.945572669452621; number of ORFs: 0.

[0610] SEQ ID NO: 170 is the sequence name of PTK7-14 v4. It is a tumor-targeting scFv construct. It is an expression codon-optimized DNA sequence encoding SEQ ID NO: 166. The sequence is: is.

[0611] SEQ ID NO: 170 includes target scFv: 1-708; target VL: 382-708; target linker: 337-381; target VH: 1-336; free energy: -352.5; gdT CAI: 0.92852740873711; number of ORFs: 0.

[0612] SEQ ID NO: 171 is the sequence name of PTK7-15. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYIMYWIRQAPGKGLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPMYTFGPGTRVEIK.

[0613] SEQ ID NO: 171 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0614] SEQ ID NO: 172 is the sequence name of PTK7-16. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYLMYWIRQAPGKGLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGIGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPMYTFGPGTRVEIK.

[0615] SEQ ID NO: 172 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 236.

[0616] SEQ ID NO: 173 is the sequence name of PTK7-17. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYLMYWIRQAPGKGLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYIAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPMYTFGPGTRVEIK.

[0617] SEQ ID NO: 173 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0618] SEQ ID NO: 174 is the sequence name of PTK7-18. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYLMYWIRQAPGKGLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSKATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPMYTFGPGTRVEIK.

[0619] SEQ ID NO: 174 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0620] SEQ ID NO: 175 is the sequence name of PTK7-19. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYLMYWIRQAPGKGLEWVSAIGSGGDTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGSLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGSSPLYTFGPGTRVEIK.

[0621] SEQ ID NO: 175 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0622] SEQ ID NO: 176 is the sequence name of SSTR2-9. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTKESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGMAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTMVTVS.

[0623] SEQ ID NO: 176 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0624] SEQ ID NO: 177 is the sequence name of SSTR2-10. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYIWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGMAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTMVTVS.

[0625] SEQ ID NO: 177 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0626] SEQ ID NO: 178 is the sequence name of SSTR2-11. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGLAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTMVTVS.

[0627] SEQ ID NO: 178 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0628] SEQ ID NO: 179 is the sequence name of SSTR2-12. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGMAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMNYWGQGTMVTVS.

[0629] SEQ ID NO: 179 includes target scFv: 1 to 248; target VL: 1 to 112; target linker: 113 to 127; and target VH: 128 to 248.

[0630] SEQ ID NO: 180 is the sequence name of SSTR2-13. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTKESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGMAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMNYWGQGTMVTVS.

[0631] SEQ ID NO: 180 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0632] SEQ ID NO: 181 is the sequence name of SSTR2-16. It is a tumor-targeting scFv construct. It is an amino acid sequence. The sequence is: EIVMTQSPDSLAVSLGERATISCKSSQSLLNSRNRKNYLAWYQQNPGQPPKLLIYWASTKESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGRLVQPGGSLRVSCEASGFTFSDYGLAWVRQAPGKGLEWVSFISNLGYSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARAPYDYDSFDPMDYWGQGTMVTVS.

[0633] SEQ ID NO: 181 includes target scFv: 1 to 236; target VL: 1 to 112; target linker: 113 to 127; target VH: 128 to 236.

[0634] SEQ ID NO: 182 is the sequence name of PTK7-17. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0635] SEQ ID NO: 182 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -363.7; gdT CAI: 0.933; and number of ORFs: 1.

[0636] SEQ ID NO: 183 is the sequence name of PTK7-19. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0637] SEQ ID NO: 183 includes target scFv: 1 to 708; target VL: 382 to 708; target linker: 337 to 381; target VH: 1 to 336; free energy: -365.5; gdT CAI: 0.933; and number of ORFs: 1.

[0638] SEQ ID NO: 184 is the sequence name of SSTR2-13. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: is.

[0639] SEQ ID NO: 184 includes target scFv: 1 to 744; target VL: 1 to 336; target linker: 337 to 381; target VH: 382 to 744; free energy: -337.6; gdT CAI: 0.904; and number of ORFs: 0.

[0640] SEQ ID NO: 185 is the sequence name of SSTR2-16. It is a tumor-targeting scFv construct. It is a DNA sequence. The sequence is: gagatcgtgatgacccagagccccgactccctggccgtttccctcggggagcgggccaccatctcctgcaagagctcccagagcctgctgaactcccggaaccggaagaactacctggcctggtaccagcagaaccccgggcagccgccgaagctgctgatctactgggcctccacgaaggagagcggggtgcccgatcgcttctccggctccggctccgggaccgacttcaccctgaccatctccagcctgcaggccgaggacgtggccgtctactactgcaagcagagctactacctctggaccttcgggcaggggaccaaggtggagatcaaaggcggcggaggctctggcggaggaggctctggaggcggcggctcccaggtgcagctggtggagagcggcggaaggctggtgcagcccggcggctccctgagggtctcctgcgaggcctccggcttcaccttctccgactacgggCtggcctgggtgaggcaggctcccgggaaggggctggagtgggtctccttcatctccaacctgggctactccatctactacgccgactccgtgaaggggcgcttcaccatctcccgggacaacgccaagaactccctctacctgcagatgaactccctgcgggccgaggacaccgccgtctactactgcgcccgggcgccctacgactacgactccttcgaccccatggactactggggccaggggaccatggtgaccgtctcc is as follows.

[0641] SEQ ID NO: 185 includes target scFv: 1 - 744; target VL: 1 - 336; target linker: 337 - 381; target VH: 382 - 744; free energy: -339.5; gdT CAI: 0.904; number of ORFs: 0.

[0642] In one variation, the disclosure provides a composition comprising an isolated polynucleotide disclosed herein as any one of SEQ ID NOs: 1-185 or encoding an amino acid sequence disclosed herein as any one of SEQ ID NOs: 1-185. Also disclosed are polynucleotides comprising the nucleotide sequence of the full-length protein of the amino acid sequence disclosed as any one of SEQ ID NOs: 1-185. Also disclosed are polynucleotides encoding proteins comprising a fragment of the amino acid sequence of any one of SEQ ID NOs: 1-185 that has biological activity, the fragment comprising 8 consecutive amino acids of any one of SEQ ID NOs: 1-185: polynucleotides that are allelic variants of the polynucleotides of any one of SEQ ID NOs: 1-185; polynucleotides that are allelic variants of the polynucleotides of any one of SEQ ID NOs: 1-185 or those disclosed above; polynucleotides that encode species homologs of the proteins encoded by any one of SEQ ID NOs: 1-185 or disclosed as any one of SEQ ID NOs: 1-185: 1-185 or any of those disclosed above; polynucleotides that hybridize under stringent conditions to any one of the polynucleotides defined by or encoded by SEQ ID NOs: 1-185 or any of those disclosed above: polynucleotides that hybridize under stringent conditions to 1-185 or any of those disclosed herein; and polynucleotides that hybridize under stringent conditions to any of the polynucleotides defined by or encoded by SEQ ID NOs: 1-185 and have a length that is at least 25% of the length of the sequence encoded by or encoded by SEQ ID NOs: 1-185.

[0643] Other systems, methods, features, and advantages of the present disclosure will be, or will become, apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this specification, be within the scope of the invention, and be protected by the following claims.

[0644] Furthermore, the present invention is not limited to the variations shown and described, as it also covers all equivalent embodiments without departing from the spirit of the present invention. Moreover, the present invention is not limited to the combination of features as described herein, but may be defined by any other combination of the individual features disclosed. Moreover, the present invention is not limited to the order of method steps as described herein, but may be defined by other combinations or orders of the disclosed steps. Anyone skilled in the art will recognize from the foregoing detailed description and any one of the figures and claims that modifications can be made to one embodiment of the disclosed invention without departing from the scope of the present invention.

Claims

1. a single chain antibody variable domain that binds to PTK7, wherein the single chain antibody variable region that binds to PTK7 has an amino acid sequence that is at least 96% identical to SEQ ID NO: 173; or a single chain antibody variable domain that binds to SSTR2, wherein the single chain antibody variable region that binds to SSTR-2 has an amino acid sequence that is at least 96% identical to SEQ ID NO: 181; A therapeutic agent comprising:

2. 2. The therapeutic agent of claim 1, further comprising, from the N-terminus to the C-terminus, a γδ T cell-optimized signal peptide that is cleaved prior to secretion, a linker, and a T cell binding protein.

3. The therapeutic agent of claim 2, wherein the linker is SEQ ID NO:

155.

4. The therapeutic agent of claim 2, wherein the T cell binding protein is SEQ ID NO:

94.

5. The method of claim 1 , wherein the single chain antibody variable domain that binds to PTK7 is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:

182.

6. The method of claim 1 , wherein the single-chain antibody variable domain that binds to SSTR2 is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:

185.

7. 1. An engineered γδ T-cell capable of secreting at least one therapeutic protein, wherein the therapeutic protein comprises, from N- to C-terminus, a γδ T-cell-optimized signal peptide, a tumor cell binding domain, a linker, and a T cell binding domain, wherein the tumor cell binding domain has an amino acid sequence at least 96% identical to SEQ ID NO: 173 or SEQ ID NO:

181.

8. 8. The engineered γδ T-cell of Claim 7, wherein the linker is SEQ ID NO:

155.

9. 9. The genetically engineered γδ T cell of claim 8, wherein the T cell binding protein is SEQ ID NO:

94.

10. 8. The engineered γδ T-cell of Claim 7, wherein the tumor cell binding domain is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:

182.

11. 8. The engineered γδ T-cell of Claim 7, wherein the tumor cell binding domain is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:

185.

12. 1. A recombinant viral vector encoding a therapeutic protein capable of biosynthesis and secretion by a γδ T cell, the therapeutic protein comprising, from N- to C-terminus, a γδ T cell-optimized signal peptide that is cleaved prior to secretion, a tumor cell binding protein domain, a linker, and a T cell binding protein, wherein the tumor cell binding protein domain is: binds to PTK7, wherein the single chain antibody variable region that binds to PTK7 is a tumor cell binding protein domain having an amino acid sequence that is at least 96% identical to SEQ ID NO: 173; or A recombinant viral vector that binds to SSTR2, wherein the single-chain antibody variable region that binds to SSTR-2 is a tumor cell binding protein domain having an amino acid sequence that is at least 96% identical to SEQ ID NO:

81.

13. 13. The recombinant viral vector of claim 12, wherein the linker is SEQ ID NO: 155 and the T cell binding protein is SEQ ID NO:

94.

14. 13. The recombinant viral vector of claim 12, wherein the tumor cell binding protein domain that binds to SSTR2 is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:

182.

15. The recombinant viral vector of claim 12, wherein the tumor cell binding protein domain that binds to GD2-3 is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:185.

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