Method for producing and using guidance and navigation control proteins

Abstract

To provide: a method for producing a therapeutic composition; and therapeutic composition.SOLUTION: A method includes: a process of providing cell materials including a cytotoxic cell; a process of incubating the cell material with first GNC protein to provide an activated cell composition, where the activated cell composition includes a first therapeutic cell; and a process of adjusting activated cell composition to provide a therapeutic composition, where the therapeutic composition is substantially free of exogenous virus and nonviral DNA or RNA. The first GNC protein includes a first cytotoxic binding site and s first cancer targeted site, where the first cytotoxic binding site has specificity to a first cytotoxic cell receptor, and is constituted so as to activate the first cytotoxic cell via bond with the first cytotoxic cell receptor, and the first cancer targeted site has specificity to the first cancer cell receptor.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62648888, filed March 27, 2018, and U.S. Provisional Patent Application No. 62648880, filed March 27, 2018, which are hereby expressly incorporated by reference in their entireties. [Technical Field]

[0002] The present application relates typically to the technical field of Guidance and Navigation Control (GNC) proteins that have multispecific binding activity to surface molecules of both immune cells and tumor cells, and more particularly to the production and use of GNC proteins, and even more particularly to the production and use of GNC proteins. [Background technology]

[0003] Cancer cells employ various strategies to evade the immune system. One mechanism underlying immune escape is impaired recognition of cancer cells by the immune system. Defective or absent presentation of cancer-specific antigens leads to immune tolerance and cancer progression. In the presence of effective immune recognition, tumors use other mechanisms to evade elimination by the immune system. Immunocompetent tumors create an inhibitory microenvironment to downregulate the immune response. Multiple players are involved in creating an inhibitory tumor microenvironment, including tumor cells, regulatory T cells, myeloid-derived suppressor cells, stromal cells, and other cell types. Suppression of the immune response can be carried out in cell-contact-dependent and cell-contact-independent ways, such as through the secretion of immunosuppressive cytokines or the removal of essential survival factors from the local environment. Cell contact-dependent inhibition relies on molecules expressed on the cell surface, such as programmed death ligand 1 (PD-L1), T-lymphocyte-associated protein 4 (CTLA-4), and other molecules (Dunn, Old et al. 2004, Adachi and Tamada 2015).

[0004] As the mechanisms by which tumors evade immune system recognition continue to be better understood, new therapies targeting these mechanisms have recently emerged. On March 25, 2011, the U.S. Food and Drug Administration (FDA) approved ipilimumab injection (Yervoy, Bristol-Myers Squibb) for the treatment of unresectable or metastatic melanoma. Yervoy binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressed on activated T cells and blocks the interaction of CTLA-4 with CD80 / 86 on antigen-presenting cells, thereby blocking the negative or inhibitory signals delivered to T cells via CTLA-4 and resulting in the reactivation of antigen-specific T cells, leading to tumor eradication in many patients. A few years later, in 2014, the FDA approved Keytruda (pembrolizumab, Merck) and Opdivo (nivolumab, Bristol-Myers Squibb) for the treatment of advanced melanoma. These monoclonal antibodies bind to PD-1 expressed on activated and / or exhausted T cells and block the interaction between PD-L1 and PD-1 expressed on tumors, thereby eliminating the PD-1-mediated inhibitory signal to T cells, resulting in the reactivation of antigen-specific T cells and, again, tumor eradication in many patients. Since then, additional clinical trials have compared the single monoclonal antibody Yervoy with the combination of the monoclonal antibodies Yervoy and Opdivo in the treatment of advanced melanoma, demonstrating improved overall survival and progression-free survival in patients treated with the antibody combination (Hodi, Chesney et al. 2016, Hellmann, Callahan et al. 2018). However, while many clinical trials have demonstrated significant benefits of treating cancer patients with monoclonal antibodies specific for one or more immune checkpoint molecules, emerging data suggest that only patients with a high mutational burden generating novel T cell epitopes recognized by antigen-specific T cells exhibit clinical responses (Snyder, Makarov et al. 2014). Patients with low tumor mutational burden rarely achieve the desired clinical response (Snyder, Makarov et al. 2014, Hellmann, Callahan et al. 2018).

[0005] In recent years, other groups have developed alternative approaches that do not require neoepitope presentation by antigen-presenting cells to activate T cells. One example is the development of bispecific antibodies. Here, the binding domain of an antibody specific for a tumor-associated antigen (e.g., CD19) is linked to an antibody binding domain specific for CD3 on T cells, thus generating a bispecific T cell engager, or BiTe, molecule. In 2014, the FDA approved a bispecific antibody called blinatumumab for the treatment of precursor B-cell acute lymphoblastic leukemia. Blinatumumab links a short-chain variable fragment (scFv) specific for CD19 expressed on leukemia cells with an scFv specific for CD3 expressed on T cells (Benjamin and Stein 2016). However, despite an initial response rate of >50% in patients with relapsed or refractory ALL, many patients are resistant to blinatumumab therapy or relapse after successful treatment with blinatumumab. Emerging evidence suggests that resistance to blinatumumab or relapse after blinatumumab treatment is due to the expression of immune checkpoint inhibitors, such as PD-L1, expressed on tumor cells, which transmit inhibitory signals via PD-1 expressed on activated T cells (Feucht, Kayser et al. 2016). In a case study of a patient who developed resistance to blinatumumab treatment, a second round of blinatumumab therapy was administered with the addition of a monoclonal antibody (pembrolizumab (Keytruda, Merck)). Blinatumumab specifically binds to PD-1, blocking the interaction between PD-1 expressed on T cells and PD-L1 expressed on tumor cells. One patient experienced a dramatic response, reducing bone marrow tumor cells from 45% to less than 5% (Feucht, Kayser et al. 2016). These results suggest that combining a bispecific BiTe molecule with one or more monoclonal antibodies can significantly improve clinical activity compared with either agent alone. Despite promising results, the cost of achieving combination therapy is expected to be high due to multiple clinical trials and difficulties in recruiting representative populations.

[0006] Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) is another promising immunotherapy for cancer treatment. Clinical success with CAR-T therapy has demonstrated durable complete remissions and prolonged survival in patients with CD19-positive, treatment-resistant B-cell malignancies (Gill and June 2015). However, the cost and complexity associated with producing personalized and genetically modified CAR-T immunotherapies has limited their production and use to specialized centers treating a relatively small number of patients. Cytokine release syndrome (CRS), also known as cytokine storm, is considered a major side effect after infusion of engineered CAR-T cells (Bonifant, Jackson et al. 2016). In many cases, the onset and severity of CRS appear to be unique to each individual patient. Current options for mitigating CRS primarily focus on rapid response and management care, due to limited options for controlling CRS before T-cell infusion.

[0007] While the efficacy of CAR-T therapy specific for CD19-positive B-cell malignancies has now been clearly established, the efficacy of CAR-T therapy for solid tumors has not yet been clearly demonstrated. Numerous clinical trials are currently underway exploring various solid tumor-associated antigens (TAAs) for CAR-T therapy. Inefficient T cell trafficking to the tumor, an immunosuppressive tumor microenvironment, suboptimal antigen recognition specificity, and lack of control of treatment-related adverse events are currently considered major obstacles to solid tumor CAR-T therapy (Li, Li et al. 2018). Options for managing not only therapeutic efficacy but also any adverse events before and after CAR-T cell infusion are limited. Summary of the Invention

[0008] The present application provides, inter alia, methods for producing therapeutic compositions comprising Guidance and Navigation Control (GNC) proteins, methods for treating cancerous conditions using Guidance and Navigation Control (GNC) proteins, and therapeutic compositions having GNC proteins or cytotoxic cells coated (or bound) with GNC proteins.

[0009] In one aspect, the present application provides a therapeutic composition. In one embodiment, the therapeutic composition comprises a cytotoxic cell, a GNC protein, and a therapeutic cell. The therapeutic cell comprises the GNC protein bound to the cytotoxic cell via a binding interaction with a cytotoxic cell receptor, and the therapeutic cell composition is substantially free of exogenous viral and non-viral DNA and RNA.

[0010] In one embodiment, the therapeutic composition further comprises a second GNC protein, a second therapeutic cell, or a combination thereof, wherein the second therapeutic cell has a cytotoxic cell to which the second GNC protein is bound, or to which both the first GNC protein and the second GNC protein are bound.

[0011] The GNC protein comprises a cytotoxic binding moiety and a cancer targeting moiety. The cytotoxic binding moiety has binding specificity for a cytotoxic cell receptor and is configured to activate cytotoxic cells upon binding to the cytotoxic cell receptor. The cancer targeting moiety has binding specificity for a cancer cell receptor.

[0012] In one embodiment, the GNC protein comprises a binding domain for a T cell receptor. Exemplary T cell receptors include, but are not limited to, CD3, CD28, PDL1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, NKp30, CD28H, CD27, CD226, CD96, CD112R, A2AR, CD160, CD244, CECAM1, CD200R, TNFRSF25 (DR3), or a combination thereof. In one embodiment, the GNC protein can activate T cells by binding a T cell binding site to a T cell receptor on the T cell. In one embodiment, the GNC protein can activate T cells by binding multiple T cell binding sites on the T cell.

[0013] In one embodiment, the GNC protein comprises a binding domain for an NK cell receptor. Exemplary NK cell receptors include, but are not limited to, receptors for activating NK cells such as CD16, NKG2D, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, NKG2C, NKG2E, and NKG2H, agonist receptors such as NKp30a, NKp30b, NKp46, NKp80, DNAM-1, CD96, CD160, 4-1BB, GITR, CD27, OX-40, and CRTAM, and antagonist receptors such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A, NKp30c, TIGIT, SIGLEC7, SIGLEC9, LILR, LAIR-1, KLRG1, PD-1, CTLA-4, and CD161.

[0014] In one embodiment, the GNC protein comprises a binding domain for a macrophage receptor. Exemplary macrophage receptors include, but are not limited to, agonist receptors on macrophages such as TLR2, TLR4, CD16, CD64, CD40, CD80, CD86, TREM-1, TREM-2, ILT-1, ILT-6a, ILT-7, ILT-8, EMR2, Dectin-1, CD69, CD32b, SIRPα, LAIR-1, VISTA, TIM-3, CD200R, and CD300a. , CD300f, SIGLEC1, SIGLEC3, SIGLEC5, SIGLEC7, SIGLEC9, ILT-2, ILT-3, ILT-4, ILT-5, LILRB3, LILRB4, DCIR, and other surface receptors such as CSF-1R, LOX-1, CCR2, FRβ, CD163, CR3, DC-SIGN, CD206, SR-A, CD36, and MARCO.

[0015] In one embodiment, the GNC protein comprises a binding domain for a dendritic cell receptor. Exemplary dendritic cell receptors include, but are not limited to, agonist receptors on dendritic cells such as TLR, CD16, CD64, CD40, CD80, CD86, HVEM, and CD70, antagonist receptors such as VISTA, TIM-3, LAG-3, and BTLA, and other surface receptors such as CSF-1R, LOX-1, CCR7, DC-SIGN, GM-CSF-R, IL-4R, IL-10R, CD36, CD206, DCIR, RIG-1, CLEC9A, and CXCR4.

[0016] In one embodiment, the GNC protein may comprise a T cell binding moiety and a cancer targeting moiety. In one embodiment, the T cell binding moiety has binding specificity for a T cell receptor including CD3, CD28, PDL1, PDL2, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, CD27, or a combination thereof. In one embodiment, the cancer targeting moiety has binding specificity for a cancer cell receptor. In one embodiment, the cancer cell receptor may include BCMA, CD19, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, a yet-to-be-discovered tumor-associated antigen, or a combination thereof.

[0017] In one embodiment, the GNC protein may have multispecific antigen binding activity for surface molecules of T cells and tumor cells. In one embodiment, the guidance and navigation control (GNC) protein comprises a binding domain for a T cell activation receptor, a binding domain for a tumor-associated antigen, a binding domain for an immune checkpoint receptor, and a binding domain for a T cell costimulatory receptor.

[0018] In one embodiment, the binding domain for the tumor-associated antigen is not adjacent to the binding domain for the T cell costimulatory receptor, hi one embodiment, the binding domain for the T cell activating receptor is adjacent to the binding domain for the tumor-associated antigen (TAA).

[0019] The T cell activation receptor may include, but is not limited to, CD3. The T cell costimulatory receptor may include, but is not limited to, 4-1BB, CD28, OX40, GITR, CD40L, ICOS, Light, CD27, CD30, or a combination thereof. The immune checkpoint receptor may include, but is not limited to, PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, or a combination thereof.

[0020] Tumor-associated antigens (TAA) may include, but are not limited to, ROR1, CD19, EGF-RVIII, BCMA, CD20, CD33, CD123, CD22, CD30, CEA, HER2, EGFR, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, or a combination thereof. In one embodiment, the tumor-associated antigen may be ROR1. In one embodiment, the tumor-associated antigen may be CD19. In one embodiment, the tumor-associated antigen may be EGF-RVIII.

[0021] In one embodiment, the guidance and navigation control (GNC) protein may be an antibody or antibody monomer or fragment thereof. In one embodiment, the GNC protein may be a triabody. In one embodiment, the GNC protein may be a tetrabody. In one embodiment, the GNC protein comprises an Fc domain or a fragment thereof. Any Fc domain from an antibody may be used. Exemplary Fc domains may include Fc domains from IgG, IgA, IgD, IgM, IgE, or fragments or combinations thereof. The Fc domain may be natural or artificial. In one embodiment, the Fc domain may comprise an antigen-binding site.

[0022] In one embodiment, the GNC protein comprises a bispecific antibody, a trispecific antibody, a tetraspecific antibody, or a combination thereof, providing up to eight binding motifs on the GNC protein. Examples of antibodies, antibody monomers, and antigen-binding fragments thereof are disclosed herein. In one embodiment, the GNC protein comprises an immunoglobulin G (IgG) moiety having two heavy chains and two light chains, with at least two scFv moieties covalently linked to either the C-terminus or N-terminus of the heavy or light chain. The IgG moiety may provide stability to the scFv moiety, and the trispecific GNC protein may have two moieties for binding to surface molecules on T cells.

[0023] In one embodiment, the guidance and navigation control (GNC) protein may be an antibody. In one embodiment, the tumor-associated antigen comprises ROR1, CD19, or EGRFVIII. In one embodiment, the T cell activating receptor comprises CD3, and the binding domain for CD3 may be linked to the binding domain for a tumor-associated antigen (TAA) via a linker to form a CD3-TAA pair. In one embodiment, an IgG Fc domain may mediate the CD3-TAA pair and the binding domain for an immune checkpoint receptor. In one embodiment, the immune checkpoint receptor may be PD-L1.

[0024] The linker may be a covalent bond or a peptide linker. In one embodiment, the peptide linker may have from about 2 to about 100 amino acid residues.

[0025] In one embodiment, the guidance and navigation control (GNC) protein has an N-terminus and a C-terminus and comprises, in tandem from the N-terminus to the C-terminus, a binding domain for CD3, a binding domain for EGF-RVIII, an IgG Fc domain, a binding domain for PD-L1, and a binding domain for 41-BB.

[0026] In one embodiment, the guidance and navigation control (GNC) protein has an N-terminus and a C-terminus and comprises, in tandem from N-terminus to C-terminus, a binding domain for 4-1BB, a binding domain for PD-L1, an IgG Fc domain, a binding domain for ROR1, and a binding domain for CD3. In one embodiment, the guidance and navigation control (GNC) protein has an N-terminus and a C-terminus and comprises, in tandem from N-terminus to C-terminus, a binding domain for CD3, a binding domain for CD19, an IgG Fc domain, a binding domain for PD-L1, and a binding domain for 4-1BB.

[0027] In one embodiment, the GNC protein comprises amino acids having a percentage homology to SEQ ID NOs: 50, 52, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110. The percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0028] In another aspect, the present application provides nucleic acid sequences encoding the disclosed GNC proteins or fragments thereof. In one embodiment, the nucleic acids have a percentage identity to SEQ ID NOs: 49, 51, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. The percentage identity is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0029] In another aspect, the present application provides a method for producing a therapeutic composition. In one embodiment, the method may include providing a cellular material containing cytotoxic cells, incubating the cellular material with a first GNC protein to provide an activated cellular composition, and preparing the activated cellular composition to provide a therapeutic composition. The activated cellular composition includes first therapeutic cells. The first therapeutic cells include a first GNC protein bound to the cytotoxic cells via a binding interaction with a first cytotoxic cell receptor. The therapeutic composition is substantially free of exogenous viruses and non-viral DNA or RNA.

[0030] In one embodiment, the cellular material comprises or is derived from PBMCs.

[0031] The first GNC protein may comprise a first cytotoxic binding moiety and a first cancer targeting moiety. The first cytotoxic binding moiety has specificity for a first cytotoxic cell receptor and is configured to activate a first cytotoxic cell through binding to the first cytotoxic cell receptor. The first cancer targeting moiety has specificity for a first cancer cell receptor.

[0032] In one embodiment, the method may repeat the incubating step by incubating the activated cell composition with a second GNC protein. The second GNC protein comprises a second cytotoxic binding moiety and a second cancer targeting moiety, the second cytotoxic binding moiety having specificity for a second cytotoxic cell receptor, and the second cancer targeting moiety having specificity for a second cancer cell receptor. The activated cell composition comprises second therapeutic cells, and the second therapeutic cells comprise the second GNC protein bound to the cytotoxic cells or the first therapeutic cells via a binding interaction with the second cytotoxic cell receptor.

[0033] In one embodiment, the first and second cancer targeting moieties independently have specificity for CD19, PDL1, or a combination thereof. In one embodiment, the first and second cytotoxic binding moieties independently have specificity for CD3, PDL1, 41BB, or a combination thereof.

[0034] The method may further include repeating the incubation by incubating the activation composition with an additional GNC protein, which may be a third GNC protein, a fourth GNC protein, etc., each providing additional therapeutic cells with the additional protein bound to the cytotoxic cell.

[0035] The first, second, and additional GNC proteins can be the same or different. A therapeutic cell can have one GNC protein, multiple identical GNC proteins, or multiple different GNC proteins bound thereto. In one embodiment, a therapeutic cell can have a first GNC protein bound thereto. In one embodiment, a therapeutic cell can have both a first and a second GNC protein bound thereto. In one embodiment, a therapeutic cell can have a first, second, and additional GNC proteins bound thereto.

[0036] In one embodiment, the therapeutic cell comprises a cytotoxic cell having at least one bound GNC protein, hi one embodiment, the therapeutic cell comprises a cytotoxic cell having at least 10, 20, 50, 100, 200, 300, 400 bound GNC proteins.

[0037] The therapeutic composition may comprise a first therapeutic cell, a first GNC protein, a cytotoxic cell, or a combination thereof. In one embodiment, the therapeutic composition may comprise a second therapeutic cell, a second GNC protein, a first therapeutic cell, a first GNC protein, a cytotoxic cell, or a combination thereof. In one embodiment, the therapeutic composition may comprise an additional GNC protein and an additional therapeutic cell.

[0038] In one embodiment, the incubating step serves to expand the therapeutic cells. In one embodiment, expanding the therapeutic cells may include incubating the therapeutic cells with an additional amount of GNC protein to provide an expanded cell population. In one embodiment, the expanded cell population has at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10The Expanded cell population comprises 4-1BB+ T cells. In one embodiment, the Expanded cell population comprises GNC-binding cells, GNC protein, cytotoxic cells, or a combination thereof. In one embodiment, to exhaust PD-1+ T cells, GNC protein may be added to the growth medium to redirect killing to PD-1+ T cells and reduce exhausted PD-1+ T cells. In one embodiment, to preferentially support PD-1+ T cells, GNC protein may be added to the growth medium to attenuate PD-1-mediated checkpoint signaling on T cells and improve PD-1+ T cell function. In one embodiment, to isolate 4-1BB-mediated costimulation via third-generation CAR-T, GNC protein may be added to the growth medium to redirect killing to 4-1BB+ T cells or to control the level of 4-1BB stimulation in therapeutic cells, such as CAR-T cells, resulting in a therapeutic composition.

[0039] In one embodiment, the cancer targeting moiety has specificity for B cells, and the therapeutic composition is substantially free of B cells. Thus, the methods disclosed herein couple activation and purification functions for therapeutic cells, thereby allowing for the production of B cell-free therapeutic compositions without the need to introduce any foreign material (such as beads) or any foreign genetic material (such as viral and non-viral DNA or RNA vectors).

[0040] In one embodiment, when the cellular material is incubated with the GNC protein, the ratio of GNC protein to cytotoxic cells is at least 30 to 1.

[0041] In one embodiment, the therapeutic composition contains at least 10 7 The cell may comprise a cell.

[0042] In a further aspect, the present application provides methods of using Guidance and Navigation Control (GNC) proteins for the treatment of cancer. In one embodiment, a method of treating a subject with cancer includes providing cytotoxic cells, conjugating the GNC protein and the cytotoxic cells to provide therapeutic cells, optionally expanding the therapeutic cells to provide an expanded cell population, and administering the therapeutic cells or expanded cell population to the subject.

[0043] In one embodiment, the method includes providing cellular material comprising cytotoxic cells, incubating the cellular material with a first GNC protein to provide an activated cellular composition, wherein the activated cellular composition comprises first therapeutic cells, preparing the activated cellular composition, providing a therapeutic composition, wherein the therapeutic composition is substantially free of exogenous viruses and non-viral DNA or RNA, and administering the therapeutic composition to a subject.

[0044] In one embodiment, the method may further comprise incubating a second GNC protein with the activated cell composition to provide an activated cell composition further comprising a second therapeutic cell. In one embodiment, the method may further comprise incubating an additional GNC protein with the activated cell composition to provide an activated cell composition further comprising an additional therapeutic cell.

[0045] In one embodiment, the method may further comprise isolating the cytotoxic cells from peripheral blood mononuclear cells (PBMCs) prior to providing the cytotoxic cells. In one embodiment, the method may further comprise isolating the peripheral blood mononuclear cells (PBMCs) from blood. In one embodiment, the blood is from the subject. In one embodiment, the blood is not from the subject. In one embodiment, the cytotoxic cells may be from the patient being treated or from another individual, such as a universal donor.

[0046] In one embodiment, the cytotoxic cells may be autologous T cells, alloreactive T cells, or universal donor T cells. In one embodiment, when autologous donor T cells are used, GNC protein may be added to the growth medium to prevent infusion of contaminating cancer cells and redirect killing to tumor antigens; exemplary tumor antigens may include CD19 for B-cell malignancies, Epcam for breast cancer, and MCP1 for melanoma.

[0047] In one embodiment, the method includes providing blood from the subject, isolating peripheral blood mononuclear cells (PBMCs) from the blood, isolating cytotoxic cells from the PBMCs, binding a GNC protein to the cytotoxic cells to provide therapeutic cells, and optionally expanding the therapeutic cells to provide an expanded cell population, and administering the therapeutic cells or expanded cell population to the subject.

[0048] In one embodiment, the method includes administering to the subject a therapeutic composition followed by administering additional GNC protein to the subject. In one embodiment, the cytotoxic cells may include CD3+ T cells, NK cells, or a combination thereof.

[0049] In one embodiment, isolating the cytotoxic cells comprises isolating at least one subpopulation of cytotoxic cells to provide therapeutic T cells. In one embodiment, the subpopulation of cytotoxic cells comprises CD4+ cells, CD8+ cells, CD56+ cells, CD69+ cells, CD107a+ cells, CD45RA+ cells, CD45RO+ cells, CD2+ cells, CD178+ cells, Granzyme+ cells, or a combination thereof.

[0050] In one embodiment, binding of the GNC protein to the cytotoxic cells comprises incubating the GNC protein with the cytotoxic cells for a period of about 2 hours to about 14 days, about 1 day to about 7 days, about 8 hours to about 24 hours, about 4 days to about 7 days, or about 10 days to about 14 days. In one embodiment, the incubation period can be greater than about 14 days. In one embodiment, the incubation period can be less than 2 hours.

[0051] In one embodiment, the ratio of GNC protein to cytotoxic cells is at least 600 to 1, 500 to 1, 400 to 1, 300 to 1, 200 to 1, 100 to 1, or 1 to 1. In one embodiment, the ratio between GNC protein and cytotoxic cells is about 1 to 1, about 10 to 1, about 100 to 1, or about 1000 to 1.

[0052] In one embodiment, the method may further comprise evaluating the efficacy of the treatment after the administration step. In one embodiment, the evaluating the efficacy of the treatment comprises checking one or more biomarkers of cancer, monitoring the lifespan of the therapeutic cells, or a combination thereof. In one embodiment, the evaluating the efficacy of the treatment comprises checking one or more biomarkers of cancer, monitoring the lifespan of the therapeutic cells, or a combination thereof. In one embodiment, the biomarkers include tumor antigens, cytokines such as gamma interferon, IL-2, IL-8, and / or chemokine release, and / or CD markers on the surface of various cell types, such as CD69, PD-1, and TIGIT, and / or tumor upon death, mutant nucleic acids released into the bloodstream by circulating tumor cells and their associated nucleic acids, or exosome-associated nucleic acids, host inflammatory mediators, or tumor-derived analytes, or a combination thereof. In one embodiment, the biomarkers include tumor antigens, tumor-associated apoptotic bodies, small molecule metabolites, cytokine release, lymphocyte surface marker expression, phosphorylated / dephosphorylated signal molecules, transcription factors, or a combination thereof.

[0053] The methods disclosed herein do not include transfecting the cytotoxic cells with a DNA or viral vector, hi one embodiment, the therapeutic cells or expanded cell population is substantially free of DNA or viral vectors.

[0054] The methods of the present invention can be used to treat a human subject suffering from cancer. In one embodiment, the cancer comprises cells expressing ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD20, CD33, CD123, CD22, CD30, CD19, an unidentified tumor-associated antigen, or a combination thereof. In one embodiment, the methods may be used to treat a mammal.

[0055] The methods disclosed herein may be used to treat a variety of cancers. Exemplary cancers include, but are not limited to, breast cancer, colon cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain cancer, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma.

[0056] In one embodiment, the method may further comprise administering an effective amount of a therapeutic agent after administering the therapeutic cells or expanded cell population to the subject. In one embodiment, the therapeutic agent comprises a monoclonal antibody, a chemotherapeutic agent, an enzyme, a protein, a costimulatory agent, or a combination thereof. In one embodiment, the costimulatory agent is configured to increase the amount of cytotoxic T cells in the subject.

[0057] The present application further provides a solution comprising an effective concentration of GNC protein. In one embodiment, the solution is plasma of a subject undergoing treatment. In one embodiment, the solution comprises GNC protein bound to cells. In one embodiment, the solution comprises GNC clusters comprising the GNC protein, T cells bound to the T cell binding domain of the GNC protein, and cancer cells bound to the cancer targeting domain of the GNC protein.

[0058] The objects and advantages of the present application will become apparent from the following detailed description of the preferred embodiment, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0059] The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, which illustrate only some embodiments prepared in accordance with the present disclosure and therefore should not be considered limiting of its scope, and the present disclosure will be explained with additional specificity and detail through the use of the accompanying drawings.

[0060] [Figure 1] FIG. 1 shows the GNC protein, which contains four antigen-specific binding domains in an antibody structure with target specificity to CD19-positive cells. [Figure 2] FIG. 2 shows that tetraspecific GNC antibodies mediate polyspecific binding between T cells and tumor cells. [Figure 3] Figure 3 is a flowchart comparing the manufacturing processes of GNC-T cell therapy (left) and CAR-T cell therapy (right). [Figure 4] FIG. 4 is a diagram illustrating sources of cellular material for preparing GNC activation therapeutic cell compositions. [Figure 5] FIG. 5 shows the selected sources of T cells for preparing GNC activating therapeutic compositions. [Figure 6] FIG. 6 shows the preparation of a T cell composition for GNC activation therapy. [Figure 7] FIG. 7 is a diagram showing the incubation and conditioning steps for preparing primary GNC-activated T cells for GNC-T cell therapy. [Figure 8] FIG. 8 shows that GNC protein (SI-35E class) induces IL-2 secretion from PBMC. [Figure 9] FIG. 9 shows that GNC protein (SI-35E class) induces granzyme B secretion from PBMC. [Figure 10] FIG. 10 shows that GNC protein (SI-35E class) induces the expression of the activation marker CD69 in CD4+ T cells. [Figure 11] FIG. 11 shows that GNC protein (SI-35E class) induces the expression of the activation marker CD69 in CD8+ T cells. [Figure 12] FIG. 12 shows that GNC protein (SI-35E class) induces the expression of the activation marker CD69 in CD56+ NK cells. [Figure 13] FIG. 13 shows that GNC proteins (SI-35E class) induce the expression of the cytotoxic degranulation marker CD107a in CD4+ T cells. [Figure 14] FIG. 14 shows that GNC protein (SI-35E class) induces the expression of the cytotoxic degranulation marker CD107a in CD8+ T cells. [Figure 15] FIG. 15 shows that GNC proteins (SI-35E class) induce the expression of the marker of cytotoxic degranulation CD107a in CD56+ NK cells. [Figure 16] FIG. 16 shows that GNC protein (SI-35E class) activates and proliferates CD3+ T cells. [Figure 17] FIG. 17 shows that GNC protein (SI-35E class) activates CD3+ T cells to secrete gamma interferon. [Figure 18] FIG. 18 shows that GNC protein (SI-35E class) activates and proliferates naive CD8+ / CD45RA+ T cells. [Figure 19] FIG. 19 shows that GNC protein (SI-35E class) activates naive CD8+ / CD45RA+ T cells to secrete gamma interferon. [Figure 20] FIG. 20 shows images of the growth of GNC-activated cells over time in 6-well G-Rex plates. [Figure 21]FIG. 21 shows an exemplary method for producing a therapeutic composition disclosed herein (A) and cell viability of PBMC, GET, and GNC-T cells after thawing (B). [Figure 22] Figure 22 shows the results of flow cytometry analysis of the PBMC-derived first GNC (SI-38E17)-activated therapeutic cell composition (Product A) (22A), the second GNC (SI-38E17)-coated therapeutic cell composition (Product B) (22B), and the input PBMC cell material (22C). [Figure 23] FIG. 23 shows the GNC-T therapeutic cell composition of GET cells and conditioned GNC-T cells from the G-Rex 100M bioreactor after thawing. [Figure 24] FIG. 24 shows the results of RTCC of CHO-ROR1 cells using GNC (SI-35E class)-coated PBMC cells. [Figure 25] FIG. 25 shows the time course kinetics of PBMC-derived SI-38E17 GNC-activated therapeutic cells for killing precursor B-cell leukemia Kasumi. [Figure 26] FIG. 26 shows the killing effect of Nalm-6, MEC-1, Daudi, and Jurkat cells using SI-38E17 GNC-activated therapeutic cells derived from PMBC. [Figure 27] FIG. 27 shows killing of Nalm-6, MEC-1, Daudi, and Jurkat leukemia cells with PBMC-derived SI-38E17 GNC-activated therapeutic cells in a spike-in model. DETAILED DESCRIPTION OF THE INVENTION

[0061] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0062] In one embodiment, guidance and navigation control (GNC) proteins are characterized by their composition of multiple antigen-specific binding domains (AgBDs) and their ability to direct T cells (or other effector cells) to cancer cells (or other target cells, such as bystander suppressor cells) through binding of multiple surface molecules on T cells and tumor cells. In one embodiment, a GNC protein is composed of Site 1 for binding at least one surface molecule on a T cell and Site 2 for binding at least one surface antigen on a cancer cell, as shown in Table 1. Figure 1 shows the structure of an exemplary tetraspecific GNC antibody comprising AgBDs for binding to both T cells expressing CD3, PD-L1, and / or 4-1BB and target B cells expressing CD19, as shown in Figure 2.

[0063] In T cell therapy, cytotoxic T cells are controlled by T cell receptor complex proteins and costimulatory signaling proteins via either agonist or antagonist receptors on their surfaces. To control this signaling and the interaction between T cells and cancer cells, multiple AgBDs may comprise Site 1 and Site 2, respectively. Examples of molecules that can be targeted by the agonistic or antagonistic binding domains of Site 1 and Site 2 are shown in Table 1. In one embodiment, the GNC protein may have at least one linker for linking Site 1 and Site 2. In one example of the GNC protein, any linker molecule can be used to link two or more AgBDs together in vitro or in vivo using a linker complementary to DNA / RNA or protein-protein interactions, including, but not limited to, biotin-avidin, leucine zipper, and any two-hybrid positive protein. In some embodiments, the linker may be an antibody backbone structure or an antibody fragment such that the GNC protein and the GNC antibody have the same meaning, for example, the structure of the exemplary tetraspecific GNC antibody in FIG. 1.

[0064] GNC proteins or antibodies can induce T cells to cancer cells in vivo or ex vivo through the binding ability of multiple AgBDs (Figure 2). The T cells may be derived from the same patient or from different individuals, and the cancer cells may be present in vivo, in vitro, or ex vivo. Examples provided herein enable the use of GNC proteins as a priming agent in T cell therapy, i.e., GNC-T cell therapy, to activate and control cytotoxic T cells ex vivo prior to adoptive transfer.

[0065] This application relates to a method for producing a GNC-activated therapeutic cell composition. Multiple AgBDs can be divided into Site 1 and Site 2, respectively, based on the interface between T cells and cancer cells (Table 1). A GNC protein with two AgBDs may simultaneously bind to a surface molecule such as CD3 on T cells and a tumor antigen such as ROR1 on tumor cells to redirect T cells to tumor cells.

[0066] The addition of a third AgBD, e.g., an AgBD that specifically binds to 41BB, can help enhance anti-CD3-induced T cell activation because 41BB is a costimulator and binding stimulates its agonist activity on activated T cells. The addition of a fourth AgBD to the GNC protein, e.g., an AgBD that specifically binds to PD-L1 on tumor cells, can block the inhibitory pathway of PD-L1 on tumor cells mediated through binding to PD-1 on T cells.

[0067] In some embodiments, these fundamental principles allow GNC proteins to be constructed to acquire multiple AgBDs for specifically binding unequal numbers of T cell antagonists and agonists to not only redirect activated T cells to tumor cells but also regulate their activity in vivo (Table 2). Thus, in some embodiments, GNC proteins may be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, or octaspecific proteins.

[0068] In one embodiment, the present application relates to GNC-T cell therapy, in which GNC protein is used to expand T cells ex vivo prior to adoptive transfer (Figure 3). Ex vivo priming of autologous T cells provides guidance and navigational control for cytotoxic T cells. For example, peripheral blood mononuclear cells (PBMCs) or specific cell types within PBMCs, such as CD8+, CD45RO+ memory T cells, can be isolated and primed ex vivo with GNC protein. These expanded cytotoxic T cells can be prepared and reinfused into patients via adoptive transfer. To control the efficacy and lifespan of cytotoxicity during in vivo cancer attack, additional GNC protein may be infused into patients. Thus, GNC-T cell therapy differs from GNC protein-based immunotherapy, in which GNC protein is administered directly to patients. However, GNC-T cell therapy does not preclude the direct administration of GNC protein to control the efficacy of infused cytotoxic T cells in a controlled manner in vivo. Additional GNC proteins can promote cytolytic activity and enhance T cell proliferation, depending on the form of AgBDs.

[0069] In one aspect, the present application relates to the production of therapeutic GNC-T cells. To compare and distinguish this process from the production of therapeutic CAR-T cells, a general process is shown in FIG. 3 for comparative purposes. In CAR-T therapy, cellular material, e.g., patient leukocytes, is collected by apheresis, a subset of CD3+ T cells is selected and activated, gene transfer is facilitated into the cellular material, and the T cell population is then expanded in number by the introduction of a foreign material scaffold for support, e.g., using anti-CD3 / anti-CD28 antibody-coated beads. Advantageously, the GNC-T cell material does not require the introduction of scaffold impurities for patient leukocyte-derived T cell expansion.

[0070] CAR-T therapy cell material must undergo gene transfer, which involves the preparation and transfection of CAR-T vector DNA, which genetically modifies the genome of T cells. These genetically modified T cells may then undergo another round of T cell expansion before being re-implanted into the patient. Random integration of CAR-T vector DNA poses the risk of T cell transformation leading to primary leukemia development, or the introduction of the CAR-T vector into leukemia cells, which increases the risk of relapse through a mechanism of internalization of the CAR target antigen (Zhang, Liu et al. 2017).

[0071] In contrast, GNC-T cell therapy has the advantage of not involving transfection with any vector DNA and therefore eliminating the risk of genetic modification prior to adoptive transfer, providing a significant advantage and technical improvement over existing CAR-T therapies. In addition to the advantage of GNC-T cell therapy being free of the risk of contamination or cancer from exogenous generic materials, the efficacy of GNC-T cell therapy may be improved if PBMCs or different T cell subsets are primed and activated ex vivo, as shown in Figures 5 and 6. A similar approach is being explored in the use of CAR-T therapy, where selected subsets of T cells can be infused back into the patient (Turtle, Hanafi et al. 2016, Turtle, Hanafi et al. 2016).

[0072] In some embodiments, it may be beneficial to remove leukemia or other cancer cells from the cellular material prior to cell expansion (Figure 7). Patient PBMCs with circulating leukemia cells, particularly from B-cell malignancies, can significantly alter their cellular composition and therefore affect the suitability of the final therapeutic cell product. For example, high levels of circulating leukemia blasts (greater than 10% of WBCs) may require depletion of leukemia cells prior to GNC-mediated cell expansion. The proportion of leukemia cells in patient-derived PBMCs may be reduced using cell fractionation methods. These methods may include steps including density gradient separation, immunofluorescence cell separation or fluorescence-activated cell sorting, immunomagnetic cell separation, or microfluidic flow chamber methods. These methods may be preceded by centrifugation, cell washing, incubation, or temperature adjustment. These methods may utilize non-cellular substrates (magnetic beads, plastics, polymers), non-cellular substrate modifications (proteins, antibodies, charge states), antibody treatment, multiplexed antibody treatment, multispecific antigen-binding proteins, and cell surface antigen-based cell coupling. These methods may use enzymatic digestion or ion chelation or mechanical agitation or cell vessel rotation. Methods for reducing leukemic blasts may utilize antibody drug conjugates or leukemia sensitizers. Methods may consist of a combination of these approaches.

[0073] In one embodiment, a tetraspecific antibody is produced and used as the GNC protein to enable the production of therapeutic T cells primed (or coated or bound) with the GNC protein. In one embodiment, the tetraspecific antibody / GNC protein comprises four different binding domains linked by antibody fragments as its backbone. One binding domain is specific for CD3 on T cells, the second binding domain is specific for a tumor-associated antigen, including but not limited to, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, and CD30, and the third and fourth binding domains are specific for two different immune checkpoint modulators, such as PD-L1, PD-L2, PD-1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, and Light.

[0074] Without being bound by theory, the advantages of GNC protein-mediated GNC-T cell therapy over conventional CAR-T therapy include, but are not limited to, the following. First, the inclusion of an IgG Fc domain confers a longer serum half-life compared to bispecific BiTe molecules. Second, the inclusion of two binding domains specific for immune checkpoint modulators can inhibit inhibitory pathways while simultaneously engaging costimulatory pathways. Third, cross-linking of tumor-associated antigens with CD3 on T cells redirects and guides T cells to kill tumor cells without the need to remove T cells from the patient and genetically modify them to be tumor-specific before reintroducing them into the patient (also known as chimeric antigen receptor T cell (CAR-T) therapy). Furthermore, fourth, GNC protein-mediated antibody therapy or T cell therapy does not involve genetic modification of T cells, which may carry the risk of clonal expansion, i.e., transformation of modified T cells into T-cell leukemia.

[0075] The disclosure herein may be more readily understood by reference to the following detailed description of specific embodiments and examples contained herein. Although the disclosure herein has been described with reference to specific details of specific embodiments thereof, such detailed description should not be construed as a limitation on the scope of the disclosure. [Example]

[0076] The following examples are offered by way of illustration and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially the same or similar results.

[0077] Example 1: GNC protein and tetraspecific GNC antibody In this application, the example of the GNC protein is a class of tetraspecific GNC antibodies, in which four AgBDs are covalently linked using an IgG antibody as the backbone (Figure 1). The first scFv from the N-terminus of this protein contains the constant domain C of the IgG antibody. H The tetraspecific GNC antibody is linked to Fab domains 1, 2, and 3, and further linked to another scFv at the C-terminus. Because each scFv domain exhibits independent binding specificity, these AgBDs do not need to be linked using the constant domains of an IgG antibody. The GNC protein structured as a tetraspecific GNC antibody directly binds to tumor-associated antigens (TAAs), engages host endogenous T cells, and kills tumor cells independently of tumor antigen presentation by MHC to antigen-specific T cell receptors (Figure 2). As shown in Figure 1, CD19 is a TAA that targets CD19-positive B cells and tumor cells. PD-L1 is an example of an immune checkpoint regulatory component of the tetraspecific GNC antibody, which can overcome the immunosuppressive tumor microenvironment and fully activate exhausted T cells within the tumor microenvironment.

[0078] Among tetraspecific GNC antibodies, the SI-35E class includes those targeting anti-human CD3 binding domains (SEQ ID NOS: 1-4), anti-human PD-L1 (SEQ ID NOS: 5-12), anti-human 4-1BB (SEQ ID NOS: 13-24), and human ROR1 (SEQ ID NOS: 25-32), i.e., TAAs. In this regard, the SI-38E and SI-39E classes target CD19 (SEQ ID NOS: 47-50) and EGFR (SEQ ID NOS: 51-54), respectively.

[0079] To construct the tetraspecific GNC antibody, AgBDs were converted into scFv and VLVH for placement in the N-terminal domain 1 (D1) of the GNC protein, or scFv and VHVL for placement in the C-terminal domains 3 (D3) and 4 (D4) of the GNC protein. All scFv molecules described here contain a flexible 20-amino acid gly-gly-gly-gly-ser (G4S) x4 linker that operably links the VH and VL, regardless of the orientation of the V region (LH or HL). The remaining domain, domain 2 (D2), of the tetraspecific GNC antibody, consists of an IgG1 heavy chain, VH-CH1-hinge-CH2-CH3, and its corresponding light chain, VL-CL, which can be either a kappa or lambda chain. D1 and D2, as well as D2, D3, and D4, are genetically linked via a 10-amino acid (G4S) x2 linker, resulting in a continuous heavy chain monomer peptide of approximately 150 kDa. When cotransfected with the appropriate light chain, the final symmetric tetraspecific GNC peptide can be purified via IgG1 Fc (Protein A / Protein G) and assayed to assess functional activity. Heavy and light chain gene "cassettes" have previously been constructed to allow for easy cloning of V-regions using either restriction enzyme sites (HindIII / NheI for heavy chain, HindIII / BsiWI for light chain) or "restriction-free cloning" tools such as Gibson Assembly (SGI-DNA, La Jolla, CA), Infusion (Takara Bio USA), or NEBuilder (NEB, Ipswich, MA); the latter was used here.

[0080] Tetraspecific GNC antibodies can be produced through a process that includes designing the intact molecule, synthesizing and cloning the nucleotide sequences of each domain, expressing them in mammalian cells, and purifying the final product. Here, the nucleotide sequences were assembled using the Geneious 10.2.3 software package (Biomatters, Auckland, NZ) and separated into their component domains for gene synthesis (Genewiz, South Plainsfield, NJ). In this example, SI-35E18 (SEQ ID NOs: 65 and 67) was separated into component domains: anti-41BB scFv VL-VH occupying D1, anti-human PD-L1 clone PL230C6 occupying D2 (Fab position), anti-human ROR1 Ig domain-specific clone 323H7 VHVL scFv occupying D3, and anti-human CD3 scFv VHVL occupying the C-terminal D4. Using the NEBuilder web-based tool, 5' and 3' nucleotides were added to each domain according to their position in the larger protein, such that each domain overlapped its neighboring domain by 20-30 nucleotides, which guided site-specific recombination. In this way, each domain could be genetically fused in a single gene assembly step. Due to the large number of homologous regions in the tetraspecific nucleotide sequence, N-terminal domains 1 and 2 were assembled separately from C-terminal domains D3 and D4. The N- and C-terminal fragments were then assembled together in a second NEBuilder reaction. A small aliquot was transformed into E. coli DH10b (Invitrogen, Carlsbad, CA), plated on TB + carbenicillin 100µg / ml plates (Teknova, Hollister, CA), and incubated overnight at 37°C. The resulting colonies were selected, and 2 mL of the overnight culture was inoculated onto TB + carbenicillin. DNA was prepared from overnight cultures (Thermo-Fisher, Carlsbad, CA) and sequenced (Genewiz, South Plainsfield, NJ) using primers flanking each domain (Sigma, St. Louis, MO). All DNA sequences were assembled and analyzed using Geneious.

[0081] Another tetraspecific GNC protein, SI-38E17 (SEQ ID NOs: 47-50), targets human CD19. The AgBDs contain anti-human 4-1BB (scFv 466F6, SEQ ID NOs: 17-20), anti-human PD-L1 (scFv PL221G5, SEQ ID NOs: 9-13), and anti-human CD3 binding domains (SEQ ID NOs: 1-4). The methods and procedures for producing this tetraspecific antibody were the same.

[0082] The GNC protein contains site 1 for binding at least one surface molecule on a T cell and site 2 for binding at least one surface antigen on a cancer cell (Table 1A). The tetraspecific GNC antibody directly engages the body's endogenous T cells and can be used to kill tumor cells independently of tumor antigen presentation by MHC to antigen-specific T cell receptors. This contrasts with therapies based solely on immune checkpoint blockade, which have been limited by antigen recognition. Depending on the situation, an immune checkpoint modulator component may be constructed as part of the tetraspecific GNC antibody, which may offer benefits similar to standard checkpoint blockade therapy.

[0083] In addition to T cells, other cytotoxic cells may also be targeted by the GNC protein for cancer killing or prevention purposes. Table 1B shows exemplary compositions of functional sites (sites 1 and 2) and antigen-binding domains in a GNC protein having an NK cell-binding domain. Table 1C shows exemplary compositions of functional sites (sites 1 and 2) and antigen-binding domains in a GNC protein having a macrophage-binding domain. Table 1D shows exemplary compositions of functional sites (sites 1 and 2) and antigen-binding domains in a GNC protein having a dendritic cell-binding domain.

[0084] GNC proteins are constructed to possess multiple specific AgBDs for binding unequal numbers of T cell antagonists and agonists. In this way, GNC proteins can redirect activated T cells to tumor cells and regulate their activity at specific levels in vivo (Table 2). Therefore, GNC proteins may be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, or even octaspecific proteins. In the present invention, three classes of tetraspecific GNC antibodies, namely, SI-39E, SI-35E, and SI-38E, were constructed to enable GNC-T cell therapy. The antibody domains and their specificities are listed in Table 3. The structures of tetraspecific GNC antibodies targeting EGFRvIII (SI-39E), ROR1 (SI-35E), and CD19 (SI-38E) are shown in Table 4.

[0085] Example 2: GNC-activated, PBMS-derived cell composition The SI-35 class antibodies listed in Table 4 were tested for their ability to induce activation and proliferation of different cell types, including CD4+ and / or CD8+ T cells and / or CD56+ natural killer cells (NK) in PBMCs. The tetraspecific GNC antibody was prepared at a 2X final concentration and titrated at 1:10 serial dilutions in 200 μl of RPMI + 10% FBS in six wells of a 96-well plate. Human PBMCs were purified by standard Ficoll density gradient from "Leukopaque," a concentrated leukapheresis product collected from normal human peripheral blood. In the final 96-well plate, PBMCs were bound to the serially titrated GNC protein by adding 100 μL of PBMCs (100,000) and 100 μL of each antibody dilution to each well of the assay. After incubating the assay plate at 37°C for approximately 72 hours, the contents of each assay well were collected, and the numbers of CD4+ T cells, CD8+ T cells, and CD56+ NK cells were analyzed by FACS. Cells were collected from each well, transferred to a new 96-well V-bottom plate, and centrifuged at 400xg for 3 minutes. The supernatant was transferred to a 96-well plate for analysis of IL-2 and granzyme B. Cells were resuspended in 200µL of 2% FBS / PBS for FACS analysis and incubated on ice for 30 minutes. The plate was centrifuged at 400xg for 3 minutes, and the supernatant was aspirated. This wash step was repeated once more, and cells were resuspended in 100µL of 2% FBS / PBS and analyzed on a BD LSR FORTESSA.

[0086] As shown in Figure 8, all SI-35E tetraspecific GNC antibodies induced IL-2 production from PBMCs, except for those in which the scFv-binding domain was replaced with FITC at position 2 (SI-35E37) and position 4 (SI-35E39). These two proteins lacked the binding domains for PD-L1 or CD3, respectively. Granzyme B secretion into the culture supernatant followed a pattern similar to that of IL-2 production, as shown in Figure 9. Furthermore, both SI-35E37 and SI-35E3 showed much lower activity in inducing cell surface expression of the activation marker CD69 in CD4+ (Figure 10), CD8+ (Figure 11), and CD56+ (Figure 12) cells in PBMC cultures. Surface expression of the cytotoxic degranulation marker CD107a (LAMP-1) was induced less consistently in CD56+ (Figure 15) but was induced by all tested GNC proteins in CD4+ (Figure 13) and CD8+ (Figure 14) cells, except for those lacking binding at positions 2 and 4. At low concentrations, three of the GNC proteins (SI-35E42, SI-35E43, and SI-35E46) induced CD69 expression on CD4+ T cells, CD8+ T cells, and CD56+ NK cells, which correlated well with the levels of IL-2 and granzyme B secretion induced by these GNC proteins (Figures 8 and 9).

[0087] The proliferation and production of gamma interferon were measured in CD3 cells stimulated for 5 days with a panel of SI-35 class antibodies. + or naive CD8 + Measured from cultures of human CD3 T cells (70,000 cells / well). + or CD8 + CD45RA+ naive T cells were purified by EasySep™ according to the manufacturer's protocol. TM Human CD3 + or naive CD8 + T cells were enriched from peripheral blood mononuclear cells from normal donors using a T cell isolation kit (StemCell Technologies). The final cell population was >98% CD3 + or CD8 + CD45RA +The cells were determined to be T cells. Proliferation in culture was measured by staining with Alamar Blue (ThermoFisher Cat. No. DAL1100) for 1 hour at 37°C, followed by reading on a Spectramax plus 384-well reader (Molecular Devices). GNC Expand CD3 + T cell proliferation was significantly correlated with CD3 T cell proliferation in GNC-free cell cultures. + This was seen as a fold increase in cell number compared to the background of T cells (Figure 16). Proliferation was induced by all constructs tested, except those lacking the CD3-binding domain. Culture supernatants were also collected from these cultures and analyzed for the presence of gamma interferon by ELISA. Gamma interferon secretion (Figure 17) was high unless the CD3 or ROR1-binding domain was modified with FITC in the GNC construct. Naive CD8 + CD45RA + T cell proliferation (Figure 18) was significantly enhanced by the addition of soluble anti-4-1BB monoclonal antibody to cultures in the absence of 4-1BB binding on GNC. + The secretion of gamma interferon from naive CD8+ T cells was more sensitive to the presence or absence of the 4-1BB binding domain than that from naive CD8+ T cells (Figure 19).

[0088] Example 3: Scale-up and preparation of the first GNC-activated therapeutic cell composition Clinically significant numbers of 10E9 GNC-activated and -coated T cells were produced after 7 days of culture. Human PBMCs were isolated from LRS cone leukocytes by standard Ficoll density gradient from Leukopaque, an enriched leukapheresis product collected from normal human peripheral blood. After collection, cells were frozen at -80°C and then thawed before culture. Growth of SI-38E17 GNC-stimulated PBMC cultures was monitored for up to 14 days using the G-Rex plate and bioreactor culture system. The culture medium consisted of RPMI 1640, 10% fetal calf serum, 1% non-essential amino acids, 1% GlutaMax, 0.6% glutamine-alanine supplement, 15 ng / ml human IL-2, and 1 nM GNC protein. Six-well G-Rex cultures tolerated seeding densities of 25–100 million PBMCs / well for 6 days, significantly exceeding the recommended amount, while a single 50% medium change on day 7 conferred tolerance to the cells within the system. Cell clustering indicated cell activation in the medium (Figure 20). At least 250 million cells from a single leukapheresis donor were seeded into two G-Rex 100M bioreactors and cultured in 1 liter of medium for 7 days. The large medium volume allowed for continued culture without the need for medium changes. The cell yield in each 100M bioreactor was 1.2–1.4 billion cells with a viability of over 88%.

[0089] Example 4: Second GNC Activation Therapeutic Cell Composition The cells from the bioreactor were collected as the first GNC-activated therapeutic cell composition, which was optionally concentrated using the LOVO automated cell processing system (Fresenius Kabi). To prepare the second GNC-activated therapeutic cell composition, one sample (Product B) was exposed to 1 nM SI-38E17, which is the same as the first GNC in this case. This can be used as a target for treating patients with CD19-positive malignant tumors (Figure 21A).

[0090] After the second concentration step (100 mL volume) during processing in the LOVO system, the second GNC-activated therapeutic cells were washed twice and then eluted in a final volume of 54 mL in a sterile processing bag. The other sample (Product A) was only exposed to the first GNC protein during the culture step and not re-exposed during processing in the LOVO system (Figure 21A). The cells were removed from the bag, mixed 1:1 with CryoStor CS10 reagent, and frozen to -80°C. The treated cells were thawed and compared to thawed, unstimulated PBMCs from the same donor before culture.

[0091] Cell viability from GNC-expanded T cell (GET) cultures was >75% and was unaffected by exposure to additional GNC reagents (GNC-T, Product B) during processing (Figure 21B). The average cell diameter increased during culture, indicating cell activation. Flow cytometry was performed on the input PBMC cell material and two post-thaw preparations using a multicolor panel of antibodies to stain for live / dead (e780), CD45, TCRα / β, CD56, CD4, CD8, CD14, TCRγ / δ, and CD20. Gating for quantification of different cell subsets for GNC-activated T cells (Product A) and additional GNC-coated GNC-T cells (Product B) is shown (Figures 22A and 22B). The proportions of each cell subpopulation were similar between Products A and B but significantly different from the proportions of input PBMCs (Figure 22C). Figure 23 summarizes the total number and proportion of each cell subpopulation. Compared to the input PBMC cell material, the total number of white blood cells increased from 250 million to 1 billion, or 4-fold, while the total number of each T cell subpopulation increased enormously: 55-fold for α / β T cells, 45-fold for CD4+ T cells, and 78-fold for CD8+ T cells. In this context, the increase in γ / δ T cells was modest at 5-fold, with TCRα / β- / lo, γ / δ+, and CD8+ T cells appearing to be most abundant. Finally, a distinctive feature of both Product A and the Product B cell composition is the absence of detectable B cells.

[0092] This example demonstrates the many advantages of GNC-T cells compared with CAR-T cell preparations. First, the starting cell composition was fresh PBMCs from donors, eliminating the need for preselection of specific cell subsets and the addition of feeder cells or synthetic beads. GNC protein is a 100% non-nucleotide biological material, eliminating the need for intracellular RNA or DNA introduction or viral vector transfection. GNC-induced expansion yielded a therapeutic dose in 9 days, compared with an average of 40 days for CAR-T cell expansion. The resulting cells were free of B cells and highly enriched in activated CD4+ and CD8+ T cells with potent killing capacity against specific targets. GNC therapeutic compositions remained viable and bioactive upon thawing from -80°C. These combined advantages are expected to significantly reduce the wait times, costs, and infrastructure- and training-related issues associated with CAR-T cell therapy. Improved purity, safety, and quantity of the final product will greatly benefit patients.

[0093] Example 5: PBMCs pre-activated with GNC protein are redirected to potently kill tumor cells Six proteins from the GNC SI-35 class, listed in Table 4, were tested for their ability to activate PBMCs for redirected T-cell cytotoxicity (RTCC) activity against a human ROR1-transfected CHO cell line (Figure 24). GNC proteins were prepared at a 2X final concentration and titrated 1:3 in 200 μl of RPMI + 10% FBS in 10 wells of a 96-well plate. In the final 96-well plate, PBMCs were bound to the serially titrated antibodies by adding 100 μL of PBMCs (200,000) and 100 μL of each antibody dilution to each well of the assay. The assay plate was incubated at 37°C for approximately 72 hours before adding CFSE-labeled CHO-ROR1 cells. 5×10e6 CHO-ROR1 target cells were labeled with 0.5 μM CFSE (Invitrogen, #C34554) in 10 mL of medium and incubated at 37°C for 20 minutes. CHO-ROR1 cells were washed three times with 50 mL of medium, resuspended in 10 mL of medium, and counted again before adding 5,000 CFSE-labeled CHO-ROR1 cells to each well of GNC-activated PBMCs. After incubating the cells for another 72 hours, the contents of each assay well were collected and analyzed for the number of remaining CFSE-labeled target cells. As shown in Figure 24, all GNC proteins tested induced RTCC activity, with SI-35E42, SI-35E43, and SI-35E46 being the most potent at reducing the number of CHO-ROR1 cells in the wells.

[0094] To further demonstrate the killing effect of GNC-labeled PBMCs against human tumor cells, we performed an escalation experiment of GNC dose and effector:target ratio using an IncuCyte S3 live cell analysis system (Sartorius / Essen Biosciences) to monitor cells over time. PBMCs from healthy donors were labeled with the GNC protein SI-38E17 at 10-fold serial doses ranging from 0.01 to 100 nM for 30 minutes at 37°C and then washed before incubation. The Kasumi-2 precursor B-cell leukemia line was selected as the target cell because the GNC protein SI-38E17 targets the CD19 antigen expressed on the B cell surface. The Kasumi-2 cells used were transfected to express green fluorescent protein (GFP), allowing the presence of tumor cells to be tracked by measuring the average green fluorescence in four images per well collected nine times over a six-day period. The effector:target (E:T) ratio was escalated by adding GNC-labeled PBMCs in serial two-fold dilutions from 5,000 (1:1) to 160,000 (32:1) cells to replicate wells. As shown in Figure 25, Kasumi-2 cells increased in number in wells with E:T ratios ranging from 1:1 to 8:1 with unlabeled PBMCs. Exposure to as little as 0.1 nM GNC resulted in increasing inhibition with each doubling of the E:T ratio, leading to decreased Kasumi-2 proliferation in 1:1 cultures. Coating PBMCs with GNC concentrations greater than 1 nM resulted in almost complete elimination of Kasumi-2 cells after 42 h of culture at all E:T ratios.

[0095] In follow-up experiments, three other transformed B cell lines, NALM-6, MEC-1, and Daud, and the acute T cell leukemia line Jurkat, were used as target cells. These target cells had previously been transfected with lentivirus to constitutively express the NucRed647 molecule. In this assay, PBMCs were exposed to a 10-fold excess of the GNC protein SI-38E17 for 30 minutes at 37°C, then washed as before. PBMCs were cultured at 1.2 x 10 6Cells were plated at 1000 cells / well and 50,000 target tumor cells were added. Cells were placed in an IncuCyte S3 set, and red fluorescent images (4 images / well) were collected at 10 time points over a 5.5-day period (Figure 26). Growth curves were established for all four tumor cell lines in the absence of PBMCs (null). Labeling PBMCs with 1 nM or more of the GNC protein SI-38E17 arrested the proliferation of all three B cell lines, but not Jurkat T cell leukemia. The B cell lines showed variable sensitivity to PBMCs pre-exposed to 0.1 nM of GNC protein.

[0096] As an alternative method for quantifying the results of culturing tumor cells and GNC-T cells, we established a limit of quantification (LOQ) curve for detection by flow cytometry. Daudi Red cells were serially diluted 10-fold, ranging from 200,000 to 20, and mixed 1:1 with 1 million PBMCs to generate samples containing 10%, 1.0%, 0.1%, 0.01%, and 0.001% tumor cells, which were analyzed by flow cytometry (Figure 27). Cells were then harvested from 15-day 6-well G-Rex cultures of 1 nM GNC-Expand T cells spiked with 10%, 1%, or 0.1% NALM-6, MEC-1, Daudi, or Jurkat tumor cells at time 0 and analyzed using the same flow cytometry settings as above. Tumor cells were reduced to less than 0.001% in all conditions except for cultures spiked with the MEC-1 tumor line at 10%, where 44 cells were detected; in this context, MEC-1 cells were reduced to <0.01% in culture.

[0097] While the disclosure herein has been described with reference to particular embodiments or examples, it may be understood that the embodiments are illustrative and that the scope of the disclosure is not so limited. Alternative embodiments of the disclosure herein may become apparent to those skilled in the art to which the disclosure herein pertains. Such alternative embodiments are deemed to be encompassed within the scope of the disclosure herein. Accordingly, the scope of the disclosure herein is defined by the appended claims and supported by the foregoing description. All references cited or mentioned in this disclosure are incorporated herein by reference in their entirety. [Table 1A] [Table 2] [Table 3] [Table 4] JPEG2024045111000006.jpg221163 JPEG2024045111000007.jpg223163 JPEG2024045111000008.jpg82163 GNC-T sequence list of tetraspecific GNC antibody The underlines in the amino acid sequence are CDRs >SEQ ID NO: 01 Anti-CD3 284A10 VHv1 nt GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACGGTGGATCATCTGCTATTACTAGTAACAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCA >SEQ ID NO: 02 Anti-CD3 284A10 VHv1 aa EVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNN IWGQGTLVTVSS >SEQ ID NO: 03 Anti-CD3 284A10 VLv1 nt GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO:04 Anti-CD3 284A10 VLv1 aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK >SEQ ID NO:05 Anti-PD-L1 PL230C6 VHv3 nt CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTACTTATAGTGGTAGTAGATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCTAGT >SEQ ID NO:06 Anti-PD-L1 PL230C6 VHv3 aa QSVEESGGGLVQPGGSLRLSCTASGIDL NTYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSS >SEQ ID NO:07 Anti-PD-L1 PL230C6 VLv2 nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 08 Anti-PD-L1 PL230C6 VLv2 aa AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNA FGGGTKVEIK >SEQ ID NO: 09 Anti-PD-L1 PL221G5 VHv1 nt GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTACGACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >SEQ ID NO: 10 Anti-PD-L1 PL221G5 VHv1 aa EVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDL WGQGTLVTVSS >SEQ ID NO: 11 Anti-PD-L1 PL221G5 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCA TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 12 Anti-PD-L1 PL221G5 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIK >SEQ ID NO: 13 Anti-4-1BB 420H5 VHv3 nt CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCAACTACTGGATATGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATGTTGGTAGTAGTGGTGACACTTACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATAGTAGTAGTTATTATATGTTTAACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >SEQ ID NO:14 Anti-4-1BB 420H5 VHv3 aa QSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSS >SEQ ID NO:15 Anti-4-1BB 420H5 VLv3 nt GCCCTTGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTGAGGACATTGATACCTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTTTATGCATCCGATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCGGTTACTATACTAGTAGTGCTGATACGAGGGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 16 Anti-4-1BB 420H5 VLv3 aa ALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK >SEQ ID NO: 17 Anti-4-1BB 466F6 VHv2 nt CGGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCATCAGTAGCTACCACATGCAGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAACCATTAGTAGTGGTGGTAATGTATACT ACGCGAGCTCCGGAGAGGCAGATTCACCATCTCCAGACCCTCGTCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTCTGGTTATAGTGATCCTATGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >SEQ ID NO: 18 Anti-4-1BB 466F6 VHv2 aa RSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSS >SEQ ID NO: 19 Anti-4-1BB 466F6 VLv5 nt GACGTTGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACCTGTCAGGCCAGTCAGAACATTAGGACTTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAGCCAATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGACCTGGAGCCTGGCGATGCTGCAACTTACTATTGTCAGTCTACCTATCTTGGTACTGATTATGTTGGCGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 20 Anti-4-1BB 466F6 VLv5 aa DVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY​​​​​​​​​​​​​EVQLLESGGGLVQPGGSLRLSCAASGIDFS RRYYMC WVRQAPGKGLEWIA CIYTGSRDTPHYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGSL WGQGTLVTVSS >SEQ ID NO: 23 Anti-4-1BB 460C3 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAGTAACTGGTTCTCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTCTGG CATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCGCAGGCGGTTACAATACTGTTATTGATACTTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 24 Anti-4-1BB 460C3 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QSSQSVYSNWFS WYQQKPGKAPKLLIY SASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC AGGYNTVIDTFA FGGGTKVEIK >SEQ ID NO: 25 Anti-ROR1 323H7 VHv4 nt GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGACATATTTATGTTAATAATGATGACACAGACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCACCTATTTCTGTGCGAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA >SEQ ID NO: 26 Anti-ROR1 323H7 VHv4 aa EVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSS >SEQ ID NO: 27 Anti-ROR1 323H7 VLv1 nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCTTCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 28 Anti-ROR1 323H7 VLv1 aa DIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIK >SEQ ID NO: 29 Anti-ROR1 338H4 VHv3 nt GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCTCCCTCAGTAGCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGATCGGAATCATTTATGCTAGTGGTAGCACAT ACTACGCGAGCTCGGCGAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAATTTATGACGGCATGGACCTCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA >SEQ ID NO: 30 Anti-ROR1 338H4 VHv3 aa EVQLVESGGGLVQPGGSLRLSCTASGFSLS SYAMS WVRQAPGRGLEWIG IIYASGSTYYASSAKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR IYDGMDL WGQGTLVTVSS >SEQ ID NO: 31 Anti-ROR1 338H4 VLv4 nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCGCCTGATCTATCTGGCATCTACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 32 Anti-ROR1 338H4 VLv4 aa DIQMTQSPSSLSASVGDRVTINC QASQNIYSYLS WYQQKPGKVPKRLIY LASTLAS GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QSNYNGNYG FGGGTKVEIK >SEQ ID NO: 33 Anti-FITC 4420 VH nt GAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGAAACAAACCTTATAATTATGAAACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGTTGAAGACATGGGTATCTATTACTGTACGGGTTCTTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA >SEQ ID NO: 34 Anti-FITC 4420 VH aa EVKLDETGGGLVQPGRPMKLSCVASGFTFS DYWMN WVRQSPEKGLEWVA QIRNKPYNYETYYSDSVKG RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTG SYYGMDY WGQGTSVTVSS >SEQ ID NO:35 Anti-FITC 4420 VL nt GATGTCGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACGTTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGGTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA >SEQ ID NO:36 Anti-FITC 4420 VL aa DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLR WYLQKPGQSPKVLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK >SEQ ID NO: 37 Human IgG1 null (G1m-fa with ADCC / CDC null mutations) nt GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT SEQ ID NO: 38 Human IgG1 null (G1m-fa with ADCC / CDC null mutations) aa ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID NO: 39 Human Ig kappa nt CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 40 Human Ig kappa aa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 41 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain nt >Sequence number 42 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain aa DIQMTQSPSTLSASVGDRVTITC QSSQSVYSNWFS WYQQKPGKAPKLLIY SASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC AGGYNTVIDTFA FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGIDFS RRYYMC WVRQAPGKGLEWIA CIYTGSRDTPHYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGSL WGQGTLVTVSSGGGGSGGGGSQSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIKGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK > SEQ ID NO: 43 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) light chain nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 44 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A...<000... AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Accession number 45 Anti-CD3 284A10 VHv1b nt<所提供的原始文本中此标签内容缺失,无法准确翻译。请补充完整后继续提问。GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGTGGTTCTTCTGCTATTACTAGTAACAACATTTGGGGCCAGGGAACCCTGGTCACCGTGTCGACA >Accession number 46 Anti-CD3 284A10 VHv1b aa EVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVST >Accession number 47 Anti-huCD19 21D4 VH nt 备注:原始文本中标签 内容缺失,无法准确翻译,请补充完整后继续提问。同时,对于一些特定的专业术语或缩写,可能需要根据具体的专利领域进行更准确的翻译调整,但在提供的文本中这些内容不太明确其确切含义,所以按照常规方式进行了翻译。GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAACCAGGAGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAGCAGTTCATGGATCGGCTGGGTGCGCCAGGCACCTGGGAAAGGCCTGGAATGGATGGGGATCATCTATCCTGATGACTCTGATACCAGATACAGTCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGGACTGCCTACCTGCAGTGGAGTAGCCTGAAGGCCTCGGACACCGCTATGTATTACTGTGCGAGACATGTTACTATGATTTGGGGAGTTATTATTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA >SEQ ID NO: 48 Anti - huCD19 21D4 VH aa EVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSS >SEQ ID NO: 49 Anti - huCD19 21D4 VL nt GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA >SEQ ID NO: 50 Anti - huCD19 21D4 VL aa AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIK >SEQ ID NO: 51 anti - huEGFRvIII 806 VH nt GATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAACTGGATTCGGCAGTTTCCAGGAAACAAGCTGGAGTGGATGGGCTACATAAGTTATAGTGGTAACACTAGGTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGCGACACATCCAAGAACCAATTCTTCCTGCAGTTGAACTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCGGGACGCGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA >SEQ ID NO: 52 anti - huEGFRvIII 806 VH aa DVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG YISYSGNTRYNPSLKS RISITRDTSKNQFFLQLNSVTIEDTATYYCVT AGRGFPY WGQGTLVTVSA >SEQ ID NO: �3 anti - huEGFRvIII 806 VL nt GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACT TGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA >SEQ ID NO: 54 Anti-huEGFRvIII 806 VL aa DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIK >SEQ ID NO: 55 GGGGSGGGGSG linker nt GGCGGTGGAGGGTCCGGCGGTGGTGGCTCCGGA >SEQ ID NO: 56 GGGGSGGGGSG linker aa GGGGSGGGGSG >SEQ ID NO: 57 GGGGSGGGGS linker 01 nt GGCGGTGGAGGGTCCGGCGGTGGTGGATCA >SEQ ID NO: 58 GGGGSGGGGS linker 01 aa GGGGSGGGGS >SEQ ID NO: 59 GGGGSGGGGS linker 02 nt GGCGGTGGAGGGTCCGGCGGTGGTGGATCC >SEQ ID NO: 60 GGGGSGGGGS linker 02 aa GGGGSGGGGS >SEQ ID NO: 61 GGGGSGGGGSGGGGSGGGGS linker nt GGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCGGCGGTGGAGGATCA >SEQ ID NO: 62 GGGGSGGGGSGGGGSGGGGS linker aa GGGGSGGGGSGGGGSGGGGS >SEQ ID NO: 63 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain nt >Accession number 64 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG YISYSGNTRYNPSLKS RISITRDTSKNQFFLQLNSVTIEDTATYYCVT AGRGFPY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY CASTLE GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSQSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK > Sequence number 65 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain nt GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 66 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain aa DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 67 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain nt >SEQ ID No. 68 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain aa DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIKGGGGSGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG YISYSGNTRYNPSLKS RISITRDTSKNQFFLQLNSVTIEDTATYYCVT AGRGFPY WGQGTLVTVSAGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY CASTLE GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSQSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK >SEQ ID NO:69 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain nt GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO:70 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 71 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain nt >Sequence number 72 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain aa DVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSQSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIKGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK > Sequence number 73 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) light chain nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 74 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-HIL1-scFv x 284A10-HIL1-scFv) light chain aa AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 75 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Sequence number 76 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSTGGGGSGGGGSQSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM [[ID=​​​​​​​​​​GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 78 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) light chain aa AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 79 SI-35E88 (284A10-L1H1-scFv x 323H7-Fab x PL230C6-H3L2-scFv x 466F6-H2L5-scFv) heavy chain nt >SEQ ID No. 80 SI-35E88 (284A10-L1H1-scFv x 323H7-Fab x PL230C6-H3L2-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSTGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSAYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNA FGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK >SEQ ID NO:81 SI-35E88 (284A10-L1H1-scFv x 323H7-Fab x PL230C6-H3L2-scFv x 466F6-H2L5-scFv) light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO:82 SI-35E88 (284A10-L1H1-scFv x 323H7-Fab x PL230C6-H3L2-scFv x 466F6-H2L5-scFv) light chain aa DIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 83 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Accession number 84 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSTGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY CASTLE GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK > Sequence number 85 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 86 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain aa DIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 87 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Accession number 88 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY CASTLE GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK >SEQ ID NO:89 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 90 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain aa AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 91 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Accession No. 92 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain aa AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK Sequence number 93 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Sequence number 94 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. 1. A method for producing a therapeutic composition, comprising: Providing cellular material comprising cytotoxic cells; incubating the cellular material with a first GNC protein to provide an activated cell composition, the activated cell composition comprising a first therapeutic cell; wherein the first GNC protein comprises a first cytotoxic binding moiety and a first cancer targeting moiety, wherein the first cytotoxic binding moiety has specificity for a first cytotoxic cell receptor and is configured to activate the first cytotoxic cell via binding to the first cytotoxic cell receptor, and wherein the first cancer targeting moiety has specificity for a first cancer cell receptor, and wherein the first therapeutic cell comprises a first GNC protein bound to a cytotoxic cell via a binding interaction with the first cytotoxic cell receptor; and preparing said activated cell composition to provide a therapeutic composition, said therapeutic composition being substantially free of exogenous viruses and non-viral DNA or RNA; A method comprising:

2. The incubating step is repeated by incubating a second GNC protein with the activated cell composition; The second GNC protein comprises a second cytotoxic binding site and a second cancer targeting site, the second cytotoxic binding site having specificity for a second cytotoxic cell receptor, and the second cancer targeting site having specificity for a second cancer cell receptor; the activated cell composition further comprises a second therapeutic cell; The second therapeutic cell comprises a second GNC protein bound to the cytotoxic cell or the first therapeutic cell via a binding interaction with the second cytotoxic cell receptor. The method of claim 1.

3. The method of claim 2, wherein the second GNC protein is the same as the first GNC protein.

4. The method of claim 2, wherein the second GNC protein is different from the first GNC protein.

5. 3. The method of claim 1 or 2, wherein the first or second cancer targeting moiety has specificity for B cells, and the therapeutic composition is substantially free of B cells.

6. The method of claim 1, wherein the cytotoxic cell receptor comprises a T cell receptor, a NK cell receptor, a macrophage receptor, a dendritic cell receptor, or a combination thereof.

7. The method of claim 1, wherein when the cellular material is incubated with the first GNC protein, the molar to cell ratio of the first GNC protein to the cytotoxic cells is at least 30 to 1.

8. The therapeutic composition has a concentration of at least 10 per ml 6 The method of claim 1, comprising a cell.

9. The method of claim 1, wherein the therapeutic composition comprises the first therapeutic cell, the first GNC protein, the cytotoxic cell, or a combination thereof.

10. The method of claim 2, wherein the therapeutic composition comprises the second therapeutic cells, the second GNC protein, the first therapeutic cells, the first GNC protein, the cytotoxic cells, or a combination thereof.

11. The method of claim 1 , wherein the cellular matter comprises PBMCs.

12. 2. The method of claim 1, wherein the first and second cancer targeting moieties independently have specificity for CD19, PDL1, or a combination thereof.

13. The method of claim 1, wherein the first and second cytotoxic binding sites independently have specificity for CD3, PDL1, 41BB, or a combination thereof.

14. 1. A method of treating a subject having cancer, comprising: Providing cellular material comprising cytotoxic cells; incubating the cellular material with a first GNC protein to provide an activated cell composition, the activated cell composition comprising a first therapeutic cell; wherein the first GNC protein comprises a first cytotoxic binding moiety and a first cancer targeting moiety, wherein the first cytotoxic binding moiety has specificity for a first cytotoxic cell receptor and is configured to activate the first cytotoxic cell via binding to the first cytotoxic cell receptor, and wherein the first cancer targeting moiety has specificity for a first cancer cell receptor, and wherein the first therapeutic cell comprises a first GNC protein bound to a cytotoxic cell via a binding interaction with the first cytotoxic cell receptor; and preparing said activated cell composition to provide a therapeutic composition, said therapeutic composition being substantially free of exogenous viruses and non-viral DNA or RNA; administering said therapeutic composition to said subject; A method comprising:

15. The incubating step is repeated by incubating a second GNC protein with the activated cell composition; wherein the second GNC protein comprises a second cytotoxic binding site and a second cancer targeting site, wherein the second cytotoxic binding site has specificity for a second cytotoxic cell receptor, and wherein the second cancer targeting site has specificity for a second cancer cell receptor; wherein the activated cell composition further comprises a second therapeutic cell; wherein the second therapeutic cell comprises a second GNC protein bound to the cytotoxic cell or the first therapeutic cell via a binding interaction with the second cytotoxic cell receptor; The method of claim 14.

16. The method of claim 14, wherein the second GNC protein is the same as the first GNC protein.

17. The method of claim 14, wherein the second GNC protein is different from the first GNC protein.

18. 16. The method of claim 14 or 15, wherein the first or second cancer targeting moiety has specificity for B cells, and the therapeutic composition is substantially free of B cells.

19. 15. The method of claim 14, further comprising the step of isolating said cytotoxic cells from peripheral blood mononuclear cells (PBMCs) prior to providing the cellular material.

20. 20. The method of claim 19, further comprising isolating the peripheral blood mononuclear cells (PBMCs) from blood.

21. 21. The method of claim 20, wherein the blood is from a subject.

22. The method of claim 20, wherein the blood is not from a subject.

23. The method of claim 14, further comprising administering additional GNC protein to the subject after administering the therapeutic composition to the subject.

24. The method of claim 14, wherein the cytotoxic cells comprise T cells, NK cells, or a combination thereof.

25. 20. The method of claim 19, wherein said isolating cytotoxic cells comprises isolating at least a subpopulation of cytotoxic cells to provide therapeutic T cells.

26. The subpopulations of cytotoxic cells are selected from the group consisting of CD3+ cells, CD4+ cells, CD8+ cells, CD56+ cells, CD28+ cells, CD69+ cells, CD107a+ cells, CD45RA+ cells, CD45RO+ cells, γδ TCR+ cells, αβ TCR+ cells, CD25+ cells, CD127 lo / - 26. The method of claim 25, comprising a CCR7+ cell, a PD-1+ cell, or a combination thereof.

27. The method of claim 14, further comprising the step of evaluating the efficacy of treatment after said administering step.

28. 27. The method of claim 26, wherein evaluating the efficacy of the treatment comprises checking one or more biomarkers of cancer, monitoring the lifespan of the therapeutic cells, or a combination thereof.

29. 29. The method of claim 28, wherein the biomarkers include tumor antigens, cytokines such as gamma interferon, IL-2, IL-8, and / or release of chemokines, and / or CD markers on the surface of various cell types, such as CD69, PD-1, TIGIT, and / or tumor upon death, mutant nucleic acids released into the bloodstream by circulating tumor cells and associated nucleic acids thereof, or exosome-associated nucleic acids, host inflammatory mediators, or tumor-derived analytes, or combinations thereof.

30. The method of claim 14, wherein the subject is a human.

31. 15. The method of claim 14, wherein the cancer comprises cells that express ROR1, CEA, HER2, EGFR, EGFRVIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, or a combination thereof.

32. 15. The method of claim 14, wherein the cancer comprises breast cancer, colon cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain cancer, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma.

33. 15. The method of claim 14, wherein the cancer is CD19 positive.

34. 15. The method of claim 14, further comprising administering an effective amount of a therapeutic agent after administering the therapeutic composition to the subject.

35. 35. The method of claim 34, wherein the therapeutic agent comprises a monoclonal antibody, a multispecific antibody, a chemotherapeutic agent, an enzyme, a protein, a costimulatory agent, an apoptosis sensitizer, a tumor vascular disorder agent, or a combination thereof.

36. 36. The method of claim 35, wherein the costimulatory agent is configured to increase the amount of cytotoxic T cells in the subject.

37. A therapeutic composition comprising a cytotoxic cell, a GNC protein, and a therapeutic cell, The GNC protein comprises a cytotoxic binding site and a cancer targeting site, the cytotoxic binding site having specificity for a cytotoxic cell receptor, the cancer targeting site having specificity for a cancer cell receptor, and the cytotoxic binding site is configured to activate the cytotoxic cell through binding to the cytotoxic cell receptor; The therapeutic cell comprises a GNC protein bound to the cytotoxic cell via a binding interaction with a cytotoxic cell receptor; and the therapeutic cell composition is substantially free of exogenous viral and non-viral DNA and RNA; Therapeutic composition.

38. 38. The therapeutic composition of claim 37, wherein said cancer targeting moiety has specificity for B cells, and said therapeutic composition is substantially free of B cells.

39. The therapeutic composition of claim 37, further comprising a second GNC protein, a second therapeutic cell, or a combination thereof, wherein the second therapeutic cell comprises a second GNC protein bound to the cytotoxic cell or the first therapeutic cell.

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