Guidance and navigation control proteins and methods for their production and use

Abstract

To provide a guidance and navigation control (GNC) protein.SOLUTION: In one embodiment, the guidance and navigation control (GNC) protein includes a binding domain to a T-cell-activated receptor, a binding domain to a tumor-associated antigen, a binding domain to an immune checkpoint receptor, and a binding domain to a T-cell co-stimulatory receptor. The binding domain to the tumor-associated antigen is not adjacent to the binding domain to the T-cell co-stimulatory receptor. In one embodiment, the binding domain to a T-cell-activated receptor is adjacent to the binding domain to the tumor-associated antigen (TAA).SELECTED DRAWING: Figure 2A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 648880, filed March 27, 2018, and U.S. Provisional Patent Application No. 62 / 648888, 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 a suppressive microenvironment to downregulate the immune response. Multiple players are involved in the formation of a suppressive 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 a cell-contact-independent manner 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, et al., 2004, Immunity, 21(2): 137-48; Adachi & Tamada, 2015, Cancer Sci., 106(8): 945-50).

[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-1 and PD-L1 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 been conducted comparing 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 et al., 2016, Lancet Oncol. 17(11):1558-1568; Hellman et al., 2018, Cancer Cell 33(5):853-861). However, numerous clinical trials have demonstrated the significant benefit of treating cancer patients with monoclonal antibodies specific for one or more immune checkpoint molecules, and emerging data suggest that only patients with a high mutational burden that generates novel T cell epitopes recognized by antigen-specific T cells exhibit clinical responses (Snyder et al., 2014, NEJM 371:2189-2199).Patients with low tumor mutation burden rarely achieve the desired clinical response (Snyder et al., 2014, NEJM 371:2189-2199; Hellman et al., 2018, Cancer Cell 33(5):853-861).

[0005] Recently, 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 an scFv specific for CD19 expressed on leukemia cells with an scFv specific for CD3 expressed on T cells (Bejnjamin and Stein 2016, Ther Adv Hematol 7(3):142-146). However, despite an initial response rate of over 50% in patients with relapsed or refractory ALL, many patients are resistant to blinatumumab therapy or relapse after successful treatment with blinatumumab. Evidence is emerging that resistance to blinatumumab or relapse after blinatumumab treatment is due to the expression of immune checkpoint inhibitor molecules expressed on tumor cells, such as PD-L1, which drives inhibitory signals via PD-1 expressed on activated T cells (Feucht et al., 2016, Oncotarget 7(47):76902-76919). In a case study of a patient who developed resistance to blinatumumab therapy, a second round of blinatumumab therapy was performed with the addition of pembrolizumab (Keytruda, Merck), a monoclonal antibody that specifically binds to PD-1 and blocks the interaction between T cell-expressed PD-1 and tumor cell-expressed PD-L1. One patient experienced a dramatic response, with bone marrow tumor cells decreasing from 45% to less than 5% (Feucht et al., 2016, Oncotarget 7(47):76902-76919). 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 using 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 & June. 2015. Immunol Rev. 263: 68-89). However, the cost and complexity associated with producing personalized and genetically modified CAR-T immunotherapies limit their production and use to specialized centers treating relatively small numbers of patients. Cytokine release syndrome (CRS), also known as cytokine storm, is the most prominent side effect following the infusion of engineered CAR-T cells (Bonifant et al., 2011, Mol Ther Oncolytics. 3: 16011). In many cases, the onset and severity of CRS appear to be a specialized and individual event. Current options for mitigating CRS focus primarily on rapid response and management care, as options for controlling CRS prior to T-cell infusion are limited.

[0007] While the efficacy of CAR-T therapy specific for CD19-positive B-cell malignancies has been 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 et al., 2018, J Hematol Oncol. 11(1):22-40). 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 a guidance and navigation control (GNC) protein that has multispecific antigen-binding activity to 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.

[0009] In one embodiment, the binding domain for the tumor-associated antigen is not adjacent to the binding domain for the T cell costimulatory receptor. In one embodiment, the binding domain for the T cell activating receptor is adjacent to the binding domain for the tumor-associated antigen (TAA). The T cell activating 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, CD160, LOX-1, siglec-15, CD47, or a combination thereof.

[0010] 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.

[0011] In one embodiment, the tumor-associated antigen may be a receptor on lung cancer cells, liver cancer cells, breast cancer cells, colon cancer cells, anal cancer cells, pancreatic cancer cells, gallbladder cancer cells, cholangiocarcinoma cells, head and neck cancer cells, nasopharyngeal cancer cells, skin cancer cells, melanoma cells, ovarian cancer cells, prostate cancer cells, urethral cancer cells, lung cancer cells, non-small cell lung cancer cells, small cell lung cancer cells, brain tumor cells, glioma cells, neuroblastoma cells, esophageal cancer cells, gastric cancer cells, liver cancer cells, kidney cancer cells, bladder cancer cells, cervical cancer cells, endometrial cancer cells, thyroid cancer cells, eye cancer cells, sarcoma cells, bone cancer cells, leukemia cells, myeloma cells, lymphoma cells, or a combination thereof. In one embodiment, the tumor-associated antigen may be a receptor on B cells.

[0012] 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.

[0013] In one embodiment, the guidance and navigation control (GNC) protein is 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.

[0014] In one embodiment, the linker may be a covalent bond. In one embodiment, the linker may be a peptide linker. In one embodiment, the peptide linker has a length not exceeding 100 amino acids. In one embodiment, the peptide linker has a length not exceeding 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In one embodiment, the peptide linker has a length not exceeding 10 amino acids. In one embodiment, the peptide linker has a length of about 2 amino acids to about 10 amino acids. In one embodiment, the peptide linker comprises 2, 5, or 10 amino acids.

[0015] 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 EGF VIII, an IgG Fc domain, a binding domain for PD-L1, and a binding domain for 41-BB. In one embodiment, the GNC protein may comprise an amino acid sequence having a percentage homology to SEQ ID NOs: 80 and 82. The percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the GNC protein is a tetraspecific antibody.

[0016] 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 GNC protein comprises an amino acid sequence having a percentage homology to SEQ ID NOs: 88 and 90. The percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the GNC protein is a tetraspecific antibody.

[0017] 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. In one embodiment, the GNC protein comprises an amino acid sequence having a percentage identity to SEQ ID NOs: 104 and 106. The percentage identity is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the GNC protein is a tetraspecific antibody.

[0018] 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, and the percentage homology is not less than 70%, 80%, 90%, 95%, 98% or 99%.

[0019] In another aspect, the present application provides a nucleic acid sequence encoding the disclosed GNC protein or a fragment thereof. In one embodiment, the nucleic acid has 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, and the percentage identity is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

[0020] In one embodiment, a guidance and navigation control (GNC) protein comprises a cytotoxic cell binding moiety and a cancer targeting moiety. Any cytotoxic cell can be a potential binding target for the disclosed GNC proteins. Examples of cytotoxic cells include, but are not limited to, T cells, NK cells, macrophages, and dendritic cells.

[0021] In one embodiment, the GNC protein comprises a T cell binding site. The T cell binding site has binding specificity 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.

[0022] In one embodiment, the GNC protein comprises an NK cell binding site, which has binding specificity for an NK cell receptor. Exemplary NK cell receptors include, but are not limited to, receptors for activation of NK cells such as CD16, NKG2D, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, NKG2C, NKG2E, NKG2H, agonist receptors such as NKp30a, NKp30b, NKp46, NKp80, DNAM-1, CD96, CD160, 4-1BB, GITR, CD27, OX-40, 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, CD161.

[0023] In one embodiment, the GNC protein comprises a macrophage-binding site, which has binding specificity 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.

[0024] In one embodiment, the GNC protein comprises a dendritic cell-binding site. The dendritic cell-binding site has binding specificity 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.

[0025] The cancer targeting moiety has binding specificity for a cancer cell receptor. Exemplary cancer cell receptors include, but are not limited to, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, or a combination thereof.

[0026] In one embodiment, the GNC protein comprises at least one T cell binding site and at least one cancer cell binding site, wherein the T cell binding site has binding specificity for a T cell receptor comprising CD3, CD28, PDL1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, CD30, CD27, or a combination thereof, and the cancer cell binding site has binding specificity for a cancer cell receptor.

[0027] 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 comprises a bispecific antibody or antibody monomer, a trispecific antibody or antibody monomer, a tetraspecific antibody or antibody monomer, an antigen-binding fragment thereof, or a combination thereof.

[0028] In one embodiment, the GNC protein may have a first portion and a second portion. In one embodiment, the first portion may include a T cell binding portion, a NK cell binding portion, a macrophage binding portion, or a dendritic cell binding portion. The second portion includes a cancer targeting portion.

[0029] The present application further provides cytotoxic cells incorporating the GNC protein disclosed herein. In one embodiment, the cytotoxicity comprises a GNC protein and a cytotoxic cell. The cytotoxic cell may be a T cell, an NK cell, a macrophage, a dendritic cell, or a combination thereof. In one embodiment, the T cell may be an autologous T cell, an allogeneic T cell, or a universal donor T cell. In one embodiment, the cytotoxic cell comprises a T cell having a T cell activation receptor and a T cell costimulatory receptor, and the GNC protein binds to the T cell through interaction with the T cell activation receptor, the T cell costimulatory receptor, or a combination thereof.

[0030] The present application further provides cancer cells incorporating the GNC protein disclosed herein. In one embodiment, the cancer cells include cancer cells bearing tumor-associated antigens, wherein the GNC protein of claim 1 is bound to the cancer cells via interaction with the tumor-associated antigen.

[0031] The present application further provides a biological complex incorporating the GNC protein disclosed herein. In one embodiment, the biological complex comprises a T cell having a T cell activation receptor and a T cell costimulatory receptor, a cancer cell having a tumor-associated antigen, and the GNC protein of claim 1, wherein the GNC protein binds to the T cell through interaction with the T cell activation receptor, the T cell costimulatory receptor, or a combination thereof, and the GNC protein binds to the cancer cell through interaction with the tumor-associated antigen.

[0032] In a further aspect, the present application provides pharmaceutical compositions useful for treating cancerous conditions. In one embodiment, the pharmaceutical composition comprises a GNC protein or cytotoxic cell disclosed herein and a pharmaceutically acceptable carrier.

[0033] In a further aspect, the present application provides methods for producing and using the disclosed GNC proteins.

[0034] In a further aspect, the present application provides a method for treating a subject having cancer. In one embodiment, the method comprises administering to the subject an effective amount of a pharmaceutical composition disclosed herein.

[0035] 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]

[0036] 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.

[0037] [Figure 1] FIG. 1 shows a general scheme of a GNC protein characterized by the composition of multiple antigen-binding domains (AgBd) and linkers. [Figure 2] Figure 2 shows examples of GNC antibodies as one embodiment of the GNC proteins disclosed herein: 2A: tetraspecific GNC antibody with EGFRvIII AgBD (SI-39E18), 2B: tetraspecific GNC antibody with ROR1 AgBD (SI-35E20), and 2C: tetraspecific GNC antibody with CD19 AgBD (SI-38E17). [Figure 3] FIG. 3 shows how the tetraspecific GNC antibody can bind to both T cells and tumor cells via multiple AgBDs. [Figure 4] FIG. 4 shows an example of tetraspecific GNC antibody binding to human ROR1 transfected CHO cells. [Figure 5] FIG. 5 shows an example of tetraspecific GNC antibodies to human 4-1BB transfected CHO cells. [Figure 6] Figure 6 shows an example of tetraspecific GNC antibodies binding to human PD-L1 transfected CHO cells. [Figure 7] FIG. 7 shows an exemplary tetraspecific GNC antibody with binding domain 323H7 specific for the Ig domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with PBMC as effector. [Figure 8]FIG. 8 shows an exemplary tetraspecific GNC antibody with binding domain 323H7 specific for the Ig domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with CD8+, CD45RO+ memory T cells as effectors. [Figure 9] FIG. 9 shows an exemplary tetraspecific GNC antibody with binding domain 323H7 specific for the Ig domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with CD8+, CD45RA+ naive T cells as effectors. [Figure 10] FIG. 10 shows an exemplary tetraspecific GNC antibody with binding domain 338H4 specific for the frizzled domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with PBMC as effector. [Figure 11] FIG. 11 shows an exemplary tetraspecific GNC antibody with binding domain 338H4 specific for the frizzled domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with CD8+, CD45RO+ memory T cells as effectors. [Figure 12] FIG. 12 shows an exemplary tetraspecific GNC antibody with binding domain 338H4 specific for the frizzled domain of ROR1 that mediates RTCC of the B-ALL cell line Kasumi2 with CD8+, CD45RA+ naive T cells as effectors. [Figure 13] FIG. 13 shows redirected pan T cell activity against the bladder cancer cell line UM-UC-3-EGFRvIII in response to treatment with EGFRvIII-targeting tetraspecific GNC antibody. [Figure 14] FIG. 14 shows the results of measuring CD8 T cell proliferation in response to treatment with EGFRvIII-targeting tetraspecific GNC antibody. [Figure 15] FIG. 15 shows the results of tracking IFNγ secretion in response to treatment with EGFRvIII-targeting tetraspecific GNC antibody. [Figure 16] FIG. 16 shows results demonstrating redirected naive T cell cytotoxicity against the bladder cancer cell line UM-UC-3-EGFRvIII. [Figure 17] FIG. 17 shows the response of PBMCs to treatment with EGFRvIII-targeting tetraspecific GNC antibodies, measuring CD8 T cell proliferation. [Figure 18] FIG. 18 shows the results of redirected pan T cell activity against the bladder cancer cell line UM-UC-3-EGFRvIII in the presence of monocytes upon treatment with EGFRvIII-targeting tetraspecific GNC antibody. [Figure 19] Figure 19 shows the functional impact of PD-L1 and 4-1BB domains on the activity of tetraspecific GNC antibody and redirected PBMC cytotoxicity against the bladder cancer cell line UM-UC-3-EGFRvIII. [Figure 20] FIG. 20 shows the results of redirected pan T cell activity against the Kasumi-2 target cell line in response to treatment with the ROR1-targeted tetraspecific GNC antibody. [Figure 21] FIG. 21 shows the results of redirected PBMC activity against the Kasimu-2 tumor cell line in response to treatment with CD19-targeted tetraspecific GNC antibody. [Figure 22] FIG. 22 shows the proliferation of CD8 T cells in response to treatment with CD19-targeted tetraspecific GNC antibody. [Figure 23] FIG. 23 shows IFNγ production by PBMCs in response to treatment with CD19-targeted tetraspecific GNC antibody. DETAILED DESCRIPTION OF THE INVENTION

[0038] 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.

[0039] This application relates to methods for producing and using GNC proteins. In one embodiment, a guidance and navigation control (GNC) protein may contain multiple antigen-specific binding domains (AgBDs) and may have the ability to direct T cells (or other effector cells) to cancer cells (or other target cells) through binding to multiple surface molecules on T cells and tumor cells (Figure 1). In one embodiment, a GNC protein may be composed of a portion 1 for binding at least one surface molecule on a T cell and a portion 2 for binding at least one surface antigen on a cancer cell (Table 1A).

[0040] In T cell therapy, cytotoxic T cells are regulated by T cell proliferation signaling and costimulatory signaling via either agonist or antagonist receptors on their surface. To regulate these signaling pathways and the interaction between T cells and cancer, multiple AgBDs may be included, each independently for portion 1 and portion 2. The GNC protein may have at least one linker connecting portion 1 and portion 2. The linkers may vary in length. In one embodiment, the linker may be a covalent bond. In one embodiment, the linker may be a peptide having about 1 to about 100 amino acid residues.

[0041] In some embodiments, any linker molecule can be used to link two or more AgBDs together, either in vitro or in vivo, using linkers complementary to DNA / RNA or protein-protein interactions, including, but not limited to, biotin-avidin, leucine-zipper, and any two-hybrid positive protein.

[0042] In some embodiments, the linker may be an antibody backbone structure or antibody fragment, such that the terms GNC protein and GNC antibody are interchangeable, as shown in FIG. 2, which illustrates an exemplary tetraspecific GNC antibody structure. In one embodiment, the GNC protein may be a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, or octaspecific protein. In one embodiment, the GNC protein may be a monoclonal antibody. In one embodiment, the GNC protein may be a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, or octaspecific monoclonal antibody. In one embodiment, the GNC protein may be a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, or octaspecific antibody.

[0043] GNC proteins or antibodies can induce T cell binding to cancer cells in vivo or ex vivo, mediated by multiple AgBDs (Figure 3). The T cells can be derived from the same patient or from different individuals, and the cancer cells can 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 therapy, to activate and control cytotoxic T cells ex vivo prior to adoptive transfer.

[0044] In addition to T cells, other cytotoxic cells can also be used by GNC proteins for the purpose of killing or preventing cancer. Table 1B shows exemplary configurations of functional sites (sites 1 and 2) and antigen-binding domains in GNC proteins having an NK cell-binding domain. Table 1C shows exemplary configurations of functional sites (sites 1 and 2) and antigen-binding domains in GNC proteins having a macrophage-binding domain. Table 1D shows exemplary configurations of functional sites (sites 1 and 2) and antigen-binding domains in GNC proteins having a dendritic cell-binding domain.

[0045] Multiple AgBDs can be divided into Site 1 and Site 2, respectively, depending on their interface with cytotoxic cells such as T cells and cancer cells (Table 1A). However, rearrangement of multiple AgBDs may also occur randomly and in unequal numbers (Table 2). GNC proteins with two AgBDs can simultaneously bind to surface molecules such as CD3 on T cells and tumor antigens such as ROR1 on tumor cells, redirecting or guiding T cells to tumor cells. Addition of a third AgBD, for example, an AgBD that specifically binds to 41BB, can help enhance anti-CD3-induced T cell activation, as 41BB is a costimulator and its binding stimulates its agonistic activity on activated T cells. Addition of a fourth AgBD, for example, an AgBD that specifically binds to PD-L1 on tumor cells, to GNC proteins can block the inhibitory pathway of PD-L1 on tumor cells mediated through binding to PD-1 on T cells. Based on these basic principles, GNC proteins can be designed and constructed to acquire multiple AgBDs for specifically binding unequal numbers of T cell antagonists and agonists, not only to redirect activated T cells to tumor cells but also to control their activity in vivo (Table 2). Thus, GNC proteins can be designed to be any multispecific protein. Table 3 provides several exemplary GNC proteins and antibodies with the specificity of the antibody-binding domain.

[0046] In one embodiment, the GNC protein may contain a multispecific antigen-binding site characterized by two functional groups. Site 1 contains multiple antigen-binding domains (AgBDs) with specificities involved in T cell activation, agonistic costimulation, and / or inhibitory antagonist activity, and Site 2 contains an antigen-binding site with at least one cancer cell-binding specificity. The GNC protein can simultaneously bind to surface molecules such as CD3 on T cells and tumor antigens such as ROR1 on tumor cells, thereby redirecting or guiding T cells to tumor cells. Adding a third binding domain to the GNC protein may help enhance CD3-induced T cell activation through direct binding to 41BB, a costimulator that exerts agonistic activity. Furthermore, adding a fourth binding domain to the GNC protein may help bind to PD-L1 on tumor cells and block the inhibitory pathway of PD-L1 on tumor cells mediated through binding to PD-1 on T cells. In some embodiments, GNC proteins acquire multiple binding capabilities to redirect activated T cells to tumor cells, and these multiple binding capabilities may help regulate T cell activation by modulating either agonistic or antagonistic activity, or both. Some binding capabilities may be similar to those of chimeric antigen receptors on CAR-T cells or bispecific antibodies such as BiTe antibodies. Without being bound by theory, through the interaction of various domains with cytotoxic cell receptors and tumor-associated antigens, GNC proteins may offer significant advantages as therapeutic agents over conventional cell-based therapeutics (such as CAR-T and antibody therapies), including, but not limited to, improved binding efficacy, optimized cell signaling and cytotoxicity, and reduced side effects, such as reduced severity of cytokine storm syndrome.

[0047] In one embodiment, the present application provides an exemplary GNC protein having four different binding domains. The GNC protein may be a "tetrabody" whose linker and backbone comprise antibody fragments. Of the four different antigen-binding domains, one specifically binds to CD3 on T cells, the second binding domain is specific for tumor-associated antigens, 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 other tumor antigens, and the third and fourth binding domains are specific for two different immune checkpoint regulators, namely, PD-L1, PD-1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, CD39, etc. Due to the diversity of their functional definitions and structures, GNC proteins are classified as a new class of immunomodulatory agents for treating cancer. Table 4 shows a list of exemplary tetraspecific GNC antibodies.

[0048] In one embodiment, GNC-mediated immunotherapy may include antibody therapy and cell therapy types, with advantages including, but not limited to, the inclusion of an IgG Fc domain, which may provide a longer serum half-life compared to bispecific BiTe molecules; second, the inclusion of two binding domains specific for immune checkpoint modulators, which may inhibit inhibitory pathways while simultaneously engaging costimulatory pathways; third, cross-linking of CD3 on T cells with tumor-associated antigens 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 specific for tumor cells before reintroducing them into the patient (also known as chimeric antigen receptor T cell (CAR-T) therapy); and 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.

[0049] The advantages of GNC protein-mediated immunotherapy over conventional immunotherapy due to the addition of one or more binding domains include, but are not limited to, the following: First, it may have a longer serum half-life compared to bispecific BiTe molecules. Second, by including two binding domains specific for immune checkpoint modulators, it may inhibit inhibitory pathways while simultaneously engaging costimulatory pathways. Third, by crosslinking tumor-associated antigens with CD3 on T cells, it 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 specific for tumor cells 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 transforming the modified T cells into clonal expansion, i.e., T-cell leukemia.

[0050] 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]

[0051] 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.

[0052] Example 1: FACS analysis of binding of tetraspecific GNC antibodies to human ROR1-transfected CHO cells The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for binding to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1. Antibodies were prepared at 2X final concentration and titrated 1:5 in 50 μl of PBS / 2% FBS across three wells of a 96-well plate. 5,000 ROR1-CHO cells in 50 μl of PBS / 2% FBS were added. The mixture was incubated on ice for 30 minutes, washed once with 200 μl of PBS / 2% FBS, and a 1:1000 dilution of stock secondary antibody, PE goat anti-human IgG Fc, was added. The mixture was incubated on ice for 30 minutes. The cells were washed twice with 200 μl of PBS / 2% FBS, resuspended in 50 μl of PBS / 2% FBS, and analyzed on a BD LSRFORTESSA. The binding profile is shown in Figure 4. The tetraspecific antibodies SI-35E18, 19, and 20, which have the 323H7 binding domain specific for the Ig domain of ROR1, showed higher binding than the tetraspecific GNC antibodies SI-3521, 22, and 23, which have the 338H4 binding domain specific for the frizzle domain of ROR1, whereas the tetraspecific GNC antibodies SI-3524, 25, and 26, which have the 330F11 binding domain specific for the kringle domain of ROR1, did not bind.

[0053] Example 2: FACS analysis of binding of tetraspecific GNC antibodies to human 41BB-transfected CHO cells The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for binding to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1. Antibodies were prepared at 2X final concentration and titrated 1:5 in 50 μl of PBS / 2% FBS across three wells of a 96-well plate. 5,000 ROR1-CHO cells in 50 μl of PBS / 2% FBS were added. The mixture was incubated on ice for 30 minutes, washed once with 200 μl of PBS / 2% FBS, and a secondary antibody, PE goat anti-human IgG Fc, was added at a 1:1000 dilution of stock. The mixture was incubated on ice for 30 minutes. The cells were washed twice with 200 μl of PBS / 2% FBS, resuspended in 50 μl of PBS / 2% FBS, and analyzed on a BD LSRFORTESSA. The binding profile is shown in Figure 5. All of the tetraspecific GNC antibodies, except for the control SI-27E12, contained the 41BB-binding domain 460C3, 420H5, or 466F6 and bound to 41BB-expressing CHO cells with different potencies.

[0054] Example 3: FACS analysis of binding of tetraspecific GNC antibodies to human PDL1-transfected CHO cells The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for binding to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1. Antibodies were prepared at 2X final concentration and titrated 1:5 across three wells of a 96-well plate in 50 μl of PBS / 2% FBS. 5,000 ROR1-CHO cells in 50 μl of PBS / 2% FBS were added. The mixture was incubated on ice for 30 minutes, washed once with 200 μl of PBS / 2% FBS, and a secondary antibody, PE goat anti-human IgG Fc, at a 1:1000 dilution of stock was added. The mixture was incubated on ice for 30 minutes. The cells were washed twice with 200 μl of PBS / 2% FBS, resuspended in 50 μl of PBS / 2% FBS, and analyzed on a BD LSRFORTESSA. The binding profile is shown in Figure 6. All tetraspecific GNC antibodies, except for the control SI-27E15, contain the same PDL1-binding domain PL230C6 and showed very similar binding intensities to PDL1-expressing CHO cells.

[0055] Example 4: Redirected T-cell cytotoxicity (RTCC) assay using peripheral blood mononuclear cells as effectors and targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human peripheral blood mononuclear cells (PBMCs) as effectors. Kasumi 2 target cells 5x10 6 Cells were labeled with 0.5 μM CFSE (Invitrogen, #C34554) in 10 ml of medium for 20 minutes at 37°C. Cells were washed three times with 50 ml of medium before resuspending in 10 ml of medium and then counting again. Antibodies were prepared at 2X final concentrations and titrated 1:3 across 10 wells of a 96-well plate in 200 μl of RPMI + 10% FBS. Human PBMCs were purified by standard Ficoll density gradient from "Leukopaque," an enriched leukapheresis product collected from normal human peripheral blood. In the final 96-well plate, 100 μl of target cells (5,000), 50 μl of PBMCs (25,000), and 100 μl of each antibody dilution were added to each well of the assay, allowing for binding of target cells, PBMCs, and serially titrated antibodies. The assay plates were incubated at 37°C for approximately 72 hours, after which the contents of each assay well were collected and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 7, the tetraspecific GNC antibodies all contain the same PDL1-binding domain PL230C6, the same ROR1-binding domain 323H7, and the same CD3-binding domain 284A10, while also containing one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific GNC antibodies exhibited greater RTCC activity compared to the controls, except for the control SI-27E12, which does not contain the 41BB-binding domain and appears to have similar efficacy to the tetraspecific GNC antibodies SI-35E18, 19, and 20.

[0056] Example 5: Redirected T cell cytotoxicity (RTCC) assay using CD8+, CD45RO+ memory T cells as effectors and targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human CD8+, CD45RO+ memory T cells as effectors. Kasumi 2 target cells 5x10 6Cells were labeled with 0.5uM CFSE (Invitrogen, #C34554) in 10ml of media for 20 minutes at 37°C. Cells were washed three times with 50ml of media before resuspending in 10ml of media and then counting again. Antibodies were prepared at 2X final concentration and titrated 1:3 in 200ul of RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RO+ memory T cells were purified using EasySep™ according to the manufacturer's protocol. TM Human memory CD8+ T cells were enriched from PBMCs from normal donors using a human memory CD8+ T cell enrichment kit (Stemcell Technologies, #19159). The final cell population was determined to be 98% CD8+, CD45RO+ T cells by FACS analysis. Target cells, T cells, and serially titrated antibodies were combined in a final 96-well plate by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ memory T cells (25,000), 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 harvested and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 8, the tetraspecific antibodies all contain the same PDL1-binding domain PL230C6, the same ROR1-binding domain 323H7, and the same CD3-binding domain 284A10, while also containing one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific antibodies exhibited greater RTCC activity than controls that did not contain one of the 41BB, PDL1, ROR1, or CD3-binding domains.

[0057] Example 6: Redirected T cell cytotoxicity (RTCC) assay using CD8+, CD45RA+ naive T cells as effectors and targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human CD8+, CD45RA+ memory T cells as effectors. Kasumi 2 target cells 5x10 6Cells were labeled with 0.5uM CFSE (Invitrogen, #C34554) in 10ml of media for 20 minutes at 37°C. Cells were washed three times with 50ml of media before resuspending in 10ml of media and then counting again. Antibodies were prepared at 2X final concentration and titrated 1:3 in 10 wells of a 96-well plate in 200ul of RPMI + 10% FBS. Human CD8+, CD45RA+ memory T cells were purified using EasySep™ according to the manufacturer's protocol. TM Human naive CD8+ T cell isolation kits (Stemcell Technologies, #19258) were used to enrich peripheral blood mononuclear cells from normal donors. The final cell population was determined to be 98% CD8+, CD45RA+ T cells by FACS analysis (data not shown). Target cells, T cells, and serially titrated antibodies were combined in a final 96-well plate by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ T cells (25,000), 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 harvested and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 9, all tetraspecific GNC antibodies contain the same PDL1-binding domain PL230C6, the same ROR1-binding domain 323H7, and the same CD3-binding domain 284A10, while also containing one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific antibodies showed greater RTCC activity than controls that did not contain one of the 41BB, PDL1, ROR1, or CD3-binding domains.

[0058] Example 7: Redirected T-cell cytotoxicity (RTCC) assay using peripheral blood mononuclear cells as effectors and targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human peripheral blood mononuclear cells (PBMCs) as effectors. Kasumi 2 target cells 5x10 6Cells were labeled with 0.5 μM CFSE (Invitrogen, #C34554) in 10 ml of medium for 20 minutes at 37°C. Cells were washed three times with 50 ml of medium before resuspending in 10 ml of medium and then counting again. Antibodies were prepared at 2X final concentrations and titrated 1:3 across 10 wells of a 96-well plate in 200 μl of RPMI + 10% FBS. 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, 100 μl of target cells (5,000), 50 μl of PBMCs (25,000), and 100 μl of each antibody dilution were added to each well of the assay, allowing for binding of target cells, PBMCs, and serially titrated antibodies. The assay plates were incubated at 37°C for approximately 72 hours, after which the contents of each assay well were collected and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 10, the tetraspecific GNC antibodies all contain the same PDL1-binding domain PL230C6, the same ROR1-binding domain 338H4, and the same CD3-binding domain 284A10, while also containing one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific GNC antibodies exhibited greater RTCC activity compared to the controls, except for the control SI-35E36, which does not contain the 41BB-binding domain and appears to have similar efficacy to the tetraspecific GNC antibodies SI-35E18, 19, and 20.

[0059] Example 8: Redirected T cell cytotoxicity (RTCC) assay using CD8+, CD45RO+ memory T cells as effectors targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human CD8+, CD45RO+ memory T cells as effectors. Kasumi 2 target cells 5x10 6Cells were labeled with 0.5uM CFSE (Invitrogen, #C34554) in 10ml of media for 20 minutes at 37°C. Cells were washed three times with 50ml of media before resuspending in 10ml of media and then counting again. Antibodies were prepared at 2X final concentration and titrated 1:3 in 200ul of RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RO+ memory T cells were purified using EasySep™ according to the manufacturer's protocol. TM Human memory CD8+ T cells were enriched from PBMCs from normal donors using a human memory CD8+ T cell enrichment kit (Stemcell Technologies, #19159). The final cell population was determined to be 98% CD8+, CD45RO+ T cells by FACS analysis (data not shown). To the final 96-well plate, 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ memory T cells (25,000), and 100 μl of each antibody dilution were added to each well of the assay, allowing for binding of target cells, T cells, and serially titrated antibodies. Assay plates were incubated at 37°C for approximately 72 hours, after which the contents of each assay well were harvested and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 11, the tetraspecific antibodies all contain the same PDL1-binding domain PL230C6, the same ROR1-binding domain 338H4, and the same CD3-binding domain 284A10, while also containing one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific antibodies exhibited greater RTCC activity than controls that did not contain one of the 41BB, PDL1, ROR1, or CD3-binding domains.

[0060] Example 9: Redirected T cell cytotoxicity (RTCC) assay using CD8+, CD45RA+ naive T cells as effectors and targeting the B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2 The tetraspecific GNC antibodies listed in Tables 3 and 4 were tested for RTCC activity against the B-ALL cell line Kasumi 2 using human CD8+, CD45RA+ memory T cells as effectors. Kasumi 2 target cells 5x10 6Cells were labeled with 0.5uM CFSE (Invitrogen, #C34554) in 10ml of media for 20 minutes at 37°C. Cells were washed three times with 50ml of media before resuspending in 10ml of media and then counting again. Antibodies were prepared at 2X final concentration and titrated 1:3 in 10 wells of a 96-well plate in 200ul of RPMI + 10% FBS. Human CD8+, CD45RA+ memory T cells were purified using EasySep™ according to the manufacturer's protocol. TM Human naive CD8+ T cells were enriched from PBMCs from normal donors using a human naive CD8+ T cell isolation kit (Stemcell Technologies, #19258). The final cell population was determined to be 98% CD8+, CD45RA+ T cells by FACS analysis. Target cells, T cells, and serially titrated antibodies were combined in a final 96-well plate by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ T cells (25,000), 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 harvested and analyzed for the remaining number of CFSE-labeled target cells. As shown in Figure 12, the tetraspecific GNC antibody contains all of the same PDL1-binding domains PL230C6, ROR1-binding domain 338H4, and CD3-binding domain 284A10, but also contains one of the 41BB-binding domains 460C3, 420H5, and 466F6. The tetraspecific antibody did not exhibit greater RTCC activity compared to controls that did not contain one of the 41BB, PDL1, ROR1, or CD3-binding domains. This is in contrast to the tetraspecific GNC antibody described in Example 6 and shown in Figure 6, which exhibited RTCC activity in CD8+, CD45RA+ naive T cells.

[0061] Example 10: Redirected panT cell cytotoxicity against bladder cancer cell line M-UC-3-EGFRvIII The set of tetraspecific GNC antibodies listed in Table 5 was evaluated for their ability to lyse the target cells UM-UC-3-EGFRvIII. TMHuman Pan T cell isolation kits (Stemcell Technologies) were used to isolate tumor cells. The UM-UC-3-EGFRvIII cell line stably expressed nuclear-localized red fluorescent protein (RFP) delivered via lentiviral transduction (Sartorius). UM-UC-3-EGFRvIII-RFP tumor cells were cocultured with pan T cells. Target cell lysis was assessed by counting the number of viable target cells remaining in culture after 96 hours of coculture with pan T cells using flow cytometry (BD LSR Fortessa). Two tetraspecific antibodies, SI-39E18 and SI-39E29, were most effective in target tumor cell lysis (Figure 13). These two molecules are composed of adjacent binding domains for CD3 and tumor antigens (Figure 5).

[0062] Example 11: CD8 T cell proliferation in response to treatment with EGFRvIII-targeted tetraspecific antibodies The set of tetraspecific GNC antibodies listed in Table 5 was evaluated for their ability to stimulate CD8 T cell proliferation in the presence of target cells UM-UC-3-EGFRvIII. PanT cells were labeled with CellTrace Violet dye (Thermo Fisher Scientific). UM-UC-3-EGFRvIII-RFP tumor cells were cocultured with panT cells. CD8 T cell proliferation was assessed using flow cytometry (BD LSRFortessa) via dilution of CellTrace Violet dye after 96 hours of coculture. Two tetraspecific GNC antibodies, SI-39E18 and SI-39E29, were most effective at stimulating CD8 T cell proliferation in the presence of target cells (Figure 14). These two molecules are composed of adjacent binding domains for CD3 and tumor antigens (Table 5). Other molecules with potent T cell stimulatory activity have structures containing adjacent CD3 and PD-L1 domains (Table 5).

[0063] Example 12: IFNγ secretion in response to treatment with EGFRvIII-targeted tetraspecific antibodies The set of tetraspecific GNC antibodies listed in Table 5 was evaluated for their ability to induce IFNγ secretion by PBMCs. PBMCs were isolated by Ficoll gradient. PBMCs were incubated with the test molecules for 96 hours. Supernatants were collected and analyzed for the presence of IFNγ using ELISA (R&D Systems) (Figure 15). The tetraspecific GNC antibodies with the strongest activity in this study all contained adjacent CD3 and PD-L1 domains (Table 5). The least active group included molecules with adjacent CD3 and tumor antigen or 4-1BB domains. The only exception from this group of tetraspecific GNC antibodies was SI-39E18, which contains adjacent CD3 and tumor antigen domains. This molecule stimulated modest IFNγ production, less than the most active group of molecules with adjacent CD3 and PD-L1 domains but more than other molecules with similar structural arrangements. Moderate IFNγ production may be beneficial for the antitumor activity of this agent.

[0064] Example 13: Cytotoxicity of redirected naive T cells against bladder cancer cell line UM-UC-3-EGFRvIII The tetraspecific GNC antibody SI-39E18 was tested for its ability to redirect naive T cells to lyse target cells UM-UC-3-EGFRvIII. Naive T cells were then transfected with EasySep TN Human naive Pan T cells were isolated using a Stemcell Technologies isolation kit. UM-UC-3-EGFRvIII-RFP tumor cells were cocultured with naive or pan T cells. Tumor cell lysis was assessed by counting RFP-labeled tumor cell nuclei. Images were acquired using an IncuCyte live cell imager (Sartorius). Antibody activity was assessed after 120 hours of incubation. Treatments were tested at a lower effector-to-target ratio of 2.5 to 1. SI-39E18 was effective in redirecting naive T cells. The EC50 was 22.08 pM for naive T cells and 0.07 pM for pan T cells (Figure 16).

[0065] Example 14: PBMC response to treatment with EGFRvIII-targeting tetraspecific GNC antibody, CD8 T cell proliferation The set of tetraspecific GNC antibodies listed in Table 1 was evaluated for their ability to induce CD8 T cell proliferation in the absence of target cells. PBMCs were labeled with CellTrace Violet dye (Thermo Fisher Scientific) and cultured with the test molecules for 96 hours. CD8 T cell proliferation was assessed by flow cytometry (BD LSRFortessa) via dilution of CellTrace Violet dye. The most effective tested molecules shared structural similarities (Figure 17). All of these molecules contain adjacent CD3 and PD-L1 domains (Table 5).

[0066] Example 15: Redirected panT cell activity against bladder cancer cell line UM-UC-3-EGFRvIII in the presence of monocytes The set of tetraspecific GNC antibodies listed in Table 5 was evaluated for their ability to lyse the target cells UM-UC-3-EGFRvIII in the presence of monocytes. TM Monocytes were isolated from PBMCs using a human monocyte isolation kit (Stemcell Technologies). UM-UC-3-EGFRvIII-RFP tumor cells were cocultured with panT cells and monocytes. Target cell lysis was assessed by counting RFP-labeled tumor cell nuclei. Images were acquired using an IncuCyte live cell imager (Sartorius). Antibody activity was assessed after 96 hours of incubation. Two tetraspecific GNC antibodies, SI-39E18 and SI-39E29, along with molecules containing adjacent CD3 and PD-L1 binding domains, were most effective in target tumor cell lysis (Figure 18) (Table 5).

[0067] Example 16: Redirected PBMC cytotoxicity against bladder cancer cell line UM-UC-3-EGFRvIII, functional activity of different 4-1BB domains, and functional impact of PD-L1 and 4-1BB domains The tetraspecific GNC antibodies listed in Table 5 were evaluated for their ability to redirect cytotoxicity against the PBMC cancer cell line UM-UC-3-EGFRvIII (UM-UC-3-EGFRvIII). UM-UC-3-EGFRvIII-RFP tumor cells were cocultured with PBMCs. Tumor cell lysis was assessed by counting RFP-labeled tumor cell nuclei. Images were acquired using an IncuCyte live cell imager (Sartorius). Antibody activity was assessed after 96 hours of incubation. Tetraspecific GNC antibodies with different 4-1BB domains, SI-39E4, SI-39E2, and SI-39E3, showed similar activity (Figure 19). Tetraspecific GNC antibodies SI-39E1 and SI-39E5, in which the PD-L1 and 4-1BB domains were replaced with silent (non-functional) FITC domains, showed reduced lytic activity. This observation confirmed the functional contribution of the 4-1BB and PD-L1 domains.

[0068] Example 17: Granzyme B production by PBMCs in response to treatment with EGFRvIII-targeting tetraspecific GNC antibodies, effect of AgBD position on EC50 values A set of tetraspecific and EGFRvIII-targeting GNC antibodies listed in Table 5 was evaluated for their ability to induce granzyme B secretion by PBMCs. PBMCs were incubated with the test molecules for 96 hours. Supernatants were collected and analyzed for the presence of granzyme B using ELISA (R&D Systems). Granzyme B levels were plotted to determine the EC50 of each tetraspecific GNC antibody. Table 6 shows the structural location of the AgBD in each tetraspecific GNC antibody. As shown in Table 6, the most active molecules in this study all contained adjacent CD3 and PD-L1 domains and 4-1BBxTAA (EGFRvIII in this experiment). Such high levels of granzyme B secretion may be undesirable because they may result in excessive in vivo cytotoxicity. In this context, the next group of molecules, SI-39E29 and SI-39E18, which showed moderate but at least 20-fold less activity, contained adjacent CD3 and TAA (EGFRvIII in this experiment).

[0069] Example 18: Redirected pan T cell activity against Kasumi-2 target cell line in response to treatment with ROR1-targeted tetraspecific GNC antibody The set of tetraspecific GNC antibodies listed in Table 7 and SI-35E20 listed in Table 4 were evaluated for their ability to lyse Kasumi-2 target cells. The Kasumi-2 cell line stably expressed green fluorescent protein (GFP) delivered via lentiviral transduction (Clontech). Kasumi-2 tumor cells were cocultured with panT cells. Target cell lysis was assessed using flow cytometry (BD LSR Fortessa) by counting the number of viable target cells remaining in culture after 96 hours of coculture with panT cells (Figure 20). SI-35E20 was characterized as shown in Figures 4-9. The results demonstrate comparable efficacy of SI-35E20-mediated redirected panT cell activity against the Kasumi-2 target cell line.

[0070] Example 19: Redirected PBMC T cell activity against the Kasumi-2 target cell line in response to treatment with CD19-targeted tetraspecific GNC antibody The set of tetraspecific GNC antibodies listed in Table 8 was evaluated for their ability to lyse target cells, Kasumi-2. Kasumi-2-GFP tumor cells were co-cultured with PBMCs. Target cell lysis was assessed using flow cytometry (BD LSR Fortescza) by counting the number of viable target cells remaining in culture after 8 days of co-culture with PBMCs (Figure 21). SI-38E17 was one of the more effective molecules in this experiment.

[0071] Example 20: CD8 T cell proliferation in response to treatment with CD19-targeted tetraspecific GNC antibody The set of tetraspecific GNC antibodies listed in Table 8 was evaluated for their ability to stimulate CD8 T cell proliferation in the presence of target cells, Kasumi-2. PBMCs were labeled with CellTrace Violet dye (Thermo Fisher Scientific). Kasumi-2-GFP tumor cells were cocultured with PBMCs. CD8 T cell proliferation was assessed using flow cytometry (BD LSR Fortessa) via dilution of CellTrace Violet dye after 8 days of coculture. Two tetraspecific GNC antibodies, SI-38E17 and SI-38E41, were most effective in stimulating CD8 T cell proliferation in the presence of target cells (Figure 22). These two molecules consist of adjacent binding domains for CD3 and tumor antigens.

[0072] Example 21: IFNγ production by PBMCs in response to treatment with a CD19-targeted tetraspecific antibody The set of tetraspecific GNC antibodies listed in Table 8 was evaluated for their ability to induce IFNγ secretion by PBMCs in the presence of target cells Kasumi-2. Target cells and PBMCs were incubated with the test molecules for 8 days. Supernatants were collected and analyzed for the presence of IFNγ using ELISA (R&D Systems). Molecules containing adjacent CD3 and PD-L1 domains were most effective at inducing IFNγ production by PBMCs, followed by antibody SI-38E5. SI-38E17 showed intermediate activity in this experiment (Figure 23).

[0073] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, polyclonal, chimeric, single-chain, bispecific or bipotent, simianized, human, and humanized antibodies, and active fragments thereof. Examples of active fragments of known antigen-binding molecules include Fab, F(ab')2, scFv, and Fv fragments, including the products of a Fab immunoglobulin expression library, and epitope-binding fragments of any of the above antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. Immunoglobulins can be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. In one embodiment, antibodies can be whole antibodies and any antigen-binding fragments derived from whole antibodies. A typical antibody typically refers to a heterotetrameric protein having two heavy (H) chains and two light (L) chains. Each heavy chain is composed of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is composed of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into hypervariable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs arranged in the following order: From the amino terminus to the carboxy terminus are FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Within the variable regions of the light and heavy chains are binding domains that interact with antigens.

[0074] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the disclosure herein may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0075] Monoclonal antibodies may also include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0076] Monoclonal antibodies can be produced using a variety of methods, including mouse hybridoma or phage display (for a review, see Siegel. Transfus. Clin. Biol. 9:15-22 (2002)), or by molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In this disclosure, antibodies were generated by immunizing rabbits with both human PD-L1 protein and cells transiently expressing human PD-L1 on their cell surface. Rabbits are known to produce antibodies with high affinity, diversity, and specificity (Weber et al. Exp. Mol. Med. 49:e305). B cells from the immunized animals were cultured in vitro and screened for the production of anti-PD-L1 antibodies. Antibody variable genes were isolated using recombinant DNA technology, and the resulting antibodies were recombinantly expressed and further screened for desired characteristics, such as the ability to inhibit binding of PD-1 to PD-L1, the ability to bind to non-human primate PD-L1, and the ability to enhance human T cell activation. This general method of antibody discovery is similar to that described by Seeber et al. PLOS One. 9:e86184 (2014).

[0077] The term "antigen- or epitope-binding portion or fragment" refers to a fragment of an antibody that is capable of binding to an antigen (in this case, PD-L1). These fragments may retain the antigen-binding function of the intact antibody as well as additional functions. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv), consisting of the VL and VH domains of a single arm of an antibody connected by a synthetic linker in a single polypeptide chain, or a Fab fragment, which is a monovalent fragment consisting of the VL, constant light (CL), VH, and constant heavy chain 1 (CH1) domains. Antibody fragments can be smaller subfragments, consisting of a single CDR domain, particularly as small as the CDR3 region from either the VL and / or VH domain (see, e.g., Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those of skill in the art. Antibody fragments can be screened for utility using the same techniques used with intact antibodies.

[0078] "Antigen- or epitope-binding fragments" can be derived from the antibodies disclosed herein by many techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments can then be collected and concentrated by membrane filtration or the like. For further description of general techniques for isolating active fragments of antibodies, see, e.g., Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0079] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is capable of cross-linking antigen.

[0080] Fab fragments may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0081] An "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three antigen-specific CDRs) may also recognize and bind antigen, although with lower affinity than the entire binding site.

[0082] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (λ), based on the amino acid sequences of their constant domains.

[0083] Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0084] A "humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulins. Additionally, framework support residues may be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio / Technology, 9:421 (1991)). In one embodiment, a "humanized antibody" may be obtained by a genetic engineering approach that allows the production of affinity-matured, human-like polyclonal antibodies in large animals, such as rabbits (see, e.g., U.S. Pat. No. 7,129,084).

[0085] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0086] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural unless the context requires otherwise.

[0087] "Isolated" refers to a biological molecule that is free from at least some of the components with which it naturally occurs. "Isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide may be prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities.

[0088] By "recombinant" is meant that the antibody is produced using recombinant nucleic acid techniques in an exogenous host cell.

[0089] The term "antigen" refers to an entity or fragment thereof that is capable of eliciting an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0090] Also, as used herein, the term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against the immunogenic agent and contributes to a human or animal immune response. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells against the administered immunogenic composition disclosed herein to alleviate or ameliorate the disorder being treated.

[0091] "Specific binding" or "binds specifically" or "specific" for a particular antigen or epitope refers to binding that is distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0092] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 This can be demonstrated by an antibody having a KD for an antigen or epitope of M or greater, where KD refers to the off-rate of a particular antibody-antigen interaction. In one embodiment, an antibody that specifically binds to an antigen can have a KD for the antigen or epitope that is 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that of a control molecule.

[0093] Specific binding to a particular antigen or epitope can also be exhibited by an antibody having a K A or K A for the antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that for a control epitope, where K A or K A refers to the association rate of a particular antibody-antigen interaction.

[0094] The "homology" between two sequences is determined by the sequence identity. When the two sequences compared to each other are different in length, the sequence identity preferably relates to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity can be conventionally determined using a computer program. Deviations that appear in the comparison of a given sequence with the above sequences disclosed herein may be caused by, for example, addition, deletion, substitution, insertion or recombination.

[0095] 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 1B] [Table 1C] [Table 1D] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] JPEG0007781243000012.jpg222126 JPEG0007781243000013.jpg217141 JPEG0007781243000014.jpg164126 Tetraspecific GNC antibody sequence listing 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-CD3 480C8 VHv1 nt GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAATCGACCTCAGTAGCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCG AGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGACGGTGGATCATCTGCTATTAATAGTAAGAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCA >SEQ ID NO: 06 Anti-CD3 480C8 VHv1 aa EVQLVESGGGLVQPGGSLRLSCAASGIDLS SNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAINSKNI WGQGTLVTVSS >SEQ ID NO: 07 Anti-CD3 480C8 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 08 Anti-CD3 480C8 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNA FGGGTKVEIK >SEQ ID NO: 09 Anti-PD-L1 PL230C6 VHv3 nt CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTACTTATAGTGGTAGTAGATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCTAGT SEQ ID NO: 10 Anti-PD-L1 PL230C6 VHv3 aa QSVEESGGGLVQPGGSLRLSCTASGIDL NTYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSS >SEQ ID NO: 11 Anti-PD-L1 PL230C6 VLv2 nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 12 Anti-PD-L1 PL230C6 VLv2 aa AYDMTQSPSSVSASVGDRVTIKCQASEDI YSFLAWY QQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNA 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<00SS32> >SEQ ID NO: 15 Anti-4-1BB 420H5 VLv3 nt<00005SS4>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 AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK >SEQ ID NO: 21 Anti-4-1BB 460C3 VHv1 nt GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAATCGACTTCAGTAGGAGATACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATATATACTGGTAGCCGCGATACTCCTCACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGAAGGTAGCCTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >SEQ ID NO: 22 Anti-4-1BB 460C3 VHv1 aa 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 324C6 VHv2 nt CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCTCCCTCAGTAGGTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTTATACTAGTGGTAGTACATGGTACGCGAGCTGGACAAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATCCTATTATGGCGGTGATAAGACTGGTTTAGGCATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA >SEQ ID NO: 26 Anti-ROR1 324C6 VHv2 nt QSLVESGGGLVQPGGSLRLSCTASGFSLS RYYMT WVRQAPGKGLEWIG TIYTSGSTWYASWTKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR SYYGGDKTGLGI WGQGTLVTVSS >SEQ ID NO: 27 Anti-ROR1 324C6 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAATCTGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 28 Anti-ROR1 324C6 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QASQSIDSWLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSAYGVSGTSSYLYT FGGGTKVEIK >SEQ ID NO: 29 Anti-ROR1 323H7 VHv4 nt GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGACATATTTATGTTAATAATGATGACACAGACTACGCG AGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCACCTATTTCTGTGCGAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA >SEQ ID NO: 30 Anti-ROR1 323H7 VHv4 aa EVQLLESGGGLVQPGGSLRLSCAAS GFTISRYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSS >SEQ ID NO: 31 Anti-ROR1 323H7 VLv1 nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCTTCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA <00​​​​​​​​​​​​​​​​​​EVQLVESGGGLVQPGGSLRLSCTASGFSL SSYAMS WVRQAPGRGLEWIG IIYASGSTYYASSAKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR IYDGMDL WGQGTLVTVSS >SEQ ID NO: 35 Anti-ROR1 338H4 VLv4 nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCGCCTGATCTATCTGGCATCTACTC TGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 36 Anti-ROR1 338H4 VLv4 aa DIQMTQSPSSLSASVGDRVTINC QASQNIYSYLS WYQQKPGKVPKRLIY LASTLAS GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QSNYNGNYG FGGGTKVEIK >SEQ ID NO: 37 Anti-ROR1 330F11 VHv1 nt GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAATAACTACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTAGTAGTGGTGCGTATACATGGTTCGCCACCTGGGCGACAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATATTCTTCTACTACTGATTGGACCTACTTTAACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA >SEQ ID NO: 38 Anti-ROR1 330F11 VHv1 aa EVQLVESGGGLVQPGGSLRLSCAASGFSLN NYWMS WVRQAPGKGLEWIG TISSGAYTWFATWATG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YSSTTDWTYFNI WGQGTLVTVSS >SEQ ID NO: 39 Anti-ROR1 330F11 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCATCCACTCTGGAATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 40 Anti-ROR1 330F11 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QASQSINNYLA WYQQKPGKAPKLLIY RASTLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSYNGVGRTA FGGGTKVEIK >SEQ ID No. 41 Anti-FITC 4-4-20 VH nt GAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGAAACAAACCTTATAATTATGAAACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGTTGAAGACATGGGTATCTATTACTGTACGGGTTCTTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA >SEQ ID No. 42 Anti-FITC 4-4-20 VH aa EVKLDETGGGLVQPGRPMKLSCVASGFTFS DYWMN WVRQSPEKGLEWVA QIRNKPYNYETYYSDSVKG RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTG SYYGMDY WGQGTSVTVSS >SEQ ID No. 43 Anti-FITC 4-4-20 VL nt GATGTCGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACGTTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGGTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA >SEQ ID NO:44 Anti-FITC 4-4-20 VL aa DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLR WYLQKPGQSPKVLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK SEQ ID NO: 45 Human IgG1 null (G1m-fa with ADCC / CDC null mutation) nt GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT SEQ ID NO: 46 Human IgG1 null (G1m-fa with ADCC / CDC null mutations) aa ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID NO: 47 Human Ig kappa nt CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 48 Human Ig kappa aa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 49 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain nt >SEQ ID NO: 50 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain aa >SEQ ID NO: 51 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) light chain nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 52 SI-35E18 (460C3-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) light chain aa AYDMTQSPSSVSASVGDRVTIKCQASEDIYSFLAWYQQKPGKAPKLLIHSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGKNNVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 53 Anti-CD3 284A10 VHv1b nt GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGTGGTTCTTCTGCTATTACTAGTAACAACATTTGGGGCCAGGGAACCCTGGTCACCGTGTCGACA >SEQ ID NO: 54 Anti-CD3 284A10 VHv1b aa EVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVST >SEQ ID NO: 55 Anti-4-1BB 466F6b VHv2 nt CGGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCATCAGTAGCTACCACATGCAGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAACCATTAGTAGTGGTGGTAATGTATACTACGCAAGCTCCGCTAGAGGCAGATTCACCATCTCCAGACCCTCGTCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTCTGGTTATAGTGATCCTATGTGGGGCCAGGGAACCCTGGTCACCGTGTCGACA >SEQ ID NO: 56 Anti-4-1BB 466F6b VHv2 aa RSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVST >SEQ ID NO: 57 Anti-PD-L1 PL230C6 VHv3b nt CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACCGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTACTTATAGTGGTAGTAGATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTGTCGACA >SEQ ID NO: 58 Anti-PD-L1 PL230C6 VHv3b aa QSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVST >SEQ ID NO: 59 Anti-huPD-L1 PL221G5 VHv1 nt GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTACGACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >SEQ ID NO: 60 Anti-huPD-L1 PL221G5 VHv1 aa EVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDL WGQGTLVTVSS >SEQ ID NO: 61 Anti-huPD-L1 PL221G5 VLv1 nt GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >SEQ ID NO: 62 Anti - huPD - L1 PL221G5 VLv1 aa DIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYY CQQGYSWGNVDNV FGGGTKVEIK >SEQ ID NO: 63 Anti - huCD19 21D4 VH nt GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAACCAGGAGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAGCAGTTCATGGATCGGCTGGGTGCGCCAGGCACCTGGGAAAGGCCTGGAATGGATGGGGATCATCTATCCTGATGACTCTGATACCAGATACAGTCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGGACTGCCTACCTGCAGTGGAGTAGCCTGAAGGCCTCGGACACCGCTATGTATTACTGTGCGAGACATGTTACTATGATTTGGGGAGTTATTATTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA >SEQ ID NO: 64 Anti - huCD19 21D4 VH aa EVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSS >SEQ ID NO: 65 anti-huCD19 21D4 VL nt GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTT TGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA >SEQ ID NO: 66 anti-huCD19 21D4 VL aa AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIK >SEQ ID NO: 67 Anti-huEGFRvIII 806 VH nt GATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAACTGGATTCGGCAGTTTCCAGGAAACAAGCTGGAGTGGATGGGCTACATAAGTTATAGTGGTAACACTAGGTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGCGACACATCCAAGAACCAATTCTTCCTGCAGTTGAACTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCGGGACGCGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA >SEQ ID NO: 68 anti-huEGFRvIII 806 VH aa DVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG<​​​​​​​​​​​>SEQ ID NO: 70 Anti-huEGFRvIII 806 VL aa DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIK >SEQ ID NO: 71 GGGGSGGGGSG linker nt GGCGGTGGAGGGTCCGGCGGTGGTGGCTCCGGA >SEQ ID NO: 72 GGGGSGGGGSG linker aa GGGGSGGGGSG >SEQ ID NO: 73 GGGGSGGGGS linker 01 nt GGCGGTGGAGGGTCCGGCGGTGGTGGATCA >SEQ ID NO: 74 GGGGSGGGGS linker 01 aa GGGGSGGGGS >SEQ ID NO: 75 GGGGSGGGGS linker 02 nt GGCGGTGGAGGGTCCGGCGGTGGTGGATCC >SEQ ID NO: 76 GGGGSGGGGS linker 02 aa GGGGSGGGGS >SEQ ID NO: 77 GGGGSGGGGSGGGGSGGGGS linker nt GGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCGGCGGTGGAGGATCA >SEQ ID NO: 78 GGGGSGGGGSGGGGSGGGGS linker aa GGGGSGGGGSGGGGSGGGGS >SEQ ID NO: 79 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain nt >Accession number 80 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS<00......​​​​​​​​​​​​​​​​WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSQSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK >SEQ ID NO: 81 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain nt GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 82 SI-39E18 (284A10-L1H1-scFv x 806-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain aa DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGSGADYSLTISSLESEDFADYYC VQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 83 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) heavy chain nt >SEQ ID NO: 84 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<​​WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSQSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK<Q >SEQ ID NO: 85 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain nt GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 86 SI-39E29 (806-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 420H5-H3L3-scFv) light chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 87 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain nt >Accession No. 88 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) heavy chain aa DVVMTQSPSSVSASVGDRVTITC<0OO0912>WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSQSVEESGGGLVQPGGSLRLSCTASGIDLN TYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTIS RYHMT WVRQAPGKGLEWIG<00OO922>RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI It should be noted that there may be an error in the "0OO0912" in the original text, which is likely to be "0000912". The above translation is based on the corrected content for better understanding. If there are specific requirements for handling such possible errors, please let me know.WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIKGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK >SEQ ID NO:89 SI−35E20 (466F6−L5H2−scFv x PL230C6−Fab x 323H7−H4L1−scFv x 284A10−H1L1−scFv) Light chain nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 90 SI-35E20 (466F6-L5H2-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 284A10-H1L1-scFv) light chain aa AYDMTQSPSSVSASVGDRVTIKC QASEDIYSFLA WYQQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 91 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) heavy chain nt >SEQ ID No. 92 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSLLA WYQQKPGKAPKLLIY<00009�5>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 WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK > Sequence number 93 SI-35E58 (284A10-L1H1-scFv x PL230C6-Fab x 323H7-H4L1-scFv x 466F6-H2L5-scFv) light chain nt GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID No. 94 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: 95 SI-35E88 (284A10-L1H1-scFv x 323H7-Fab x PL230C6-H3L2-scFv x 466F6-H2L5-scFv) heavy chain nt >SEQ ID No. 96 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[[ID=​​​​​​GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 98 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: 99 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Sequence number 100 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 [[ID=​​​​​​​​​​​​​WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY CASTLE GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK > Sequence number 101 SI-35E99 (284A10-L1H1-scFv x 323H7-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 102 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: 103 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >Accession number 104 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK >SEQ ID NO: 105 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID NO: 106 SI-38E17 (284A10-L1H1-scFv x 21D4-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain aa AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 107 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) heavy chain nt >SEQ ID NO: 108 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 109 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL221G5-H1L1-scFv x 466F6-H2L5-scFv) light chain nt GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT SEQ ID NO: 110 SI-38E33 (21D4-LH-scFv x 284A10-Fab x PL2(21G5-H1L1-scFv x 466F6-H2L5-scFv) light chain aa DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS It should be noted that there may be some inaccuracies in the translation of the specific biological sequence terms, and it is recommended to consult relevant biological and medical experts for more accurate verification in the context of biological research.FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, a binding domain for EGFR VIII, a binding domain for CD3, IgG Fc domain, a binding domain for PD-L1, and 4-1BB binding domain A guidance and navigation control (GNC) protein comprising: In the GNC protein, the binding domain against EGFR VIII comprises VH-CDR1: SDFAWN, VH-CDR2: YISSYSGNTRYNPSLKS and VH-CDR3: AGRGFPY, and VL-CDR1: HSSQDINSNIG, VL-CDR2: HGTNLDD and VL-CDR3: VQYAQFPWT; the CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS; the binding domain against PD-L1 comprises VH-CDR1: SGYDMC, VH-CDR2: CIAAGSAGITYDANWAKG, and VH-CDR3: SAFSFDYAMDL, and VL-CDR1: QASQSISSSHLN, VL-CDR2: KASTLAS, and VL-CDR3: CQQGYSWGNVDNV; The binding domain for 4-1BB comprises VH-CDR1: SNYWIC, VH-CDR2: CIYVGSSGDTYYASSAKG and VH-CDR3: DSSSYYMFNL, and VL-CDR1: QASEDIDTYLA, VL-CDR2: YASDLAS and VL-CDR3: QGGYYTSSADTRGA. Guidance and navigation control (GNC) proteins.

2. A guidance and navigation control (GNC) protein as described in claim 1, comprising a chain of amino acid sequences shown in SEQ ID NO: 84 and a chain of amino acid sequences shown in SEQ ID NO:

86.

3. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, a binding domain for 4-1BB, a binding domain for PD-L1, IgG Fc domain, a binding domain for ROR1, and CD3 binding domain A guidance and navigation control (GNC) protein comprising: In the GNC protein, The binding domain for 4-1BB comprises VH-CDR1: RRYYMC, VH-CDR2: CIYTGSRDTPHYASSAKG and VH-CDR3: EGSL, and VL-CDR1: QSSQSVYSNWFS, VL-CDR2: SASTLAS and VL-CDR3: AGGYNTVIDTFA; the binding domain against PD-L1 comprises VH-CDR1: NTYDMI, VH-CDR2: IITYSGSRYYANWAKG, and VH-CDR3: DYMSGSHL, and VL-CDR1: YSFLAWY, VL-CDR2: SASSLAS, and VL-CDR3: QQGYGKNNVDNA; the binding domain for ROR1 comprises VH-CDR1: GFTISRYHMT, VH-CDR2: HIYVNNDDTDYASSAKG, and VH-CDR3: LDVGGGGAYIGDI, and VL-CDR1: QSSQSVYNNNDLA, VL-CDR2: YASTLAS, and VL-CDR3: AGGYDTDGLDTFA; The CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS. Guidance and navigation control (GNC) proteins.

4. A guidance and navigation control (GNC) protein as described in claim 3, comprising a chain of amino acid sequences shown in SEQ ID NO: 50 and a chain of amino acid sequences shown in SEQ ID NO:

52.

5. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, CD3 binding domain a binding domain for PD-L1, IgG Fc domain, a binding domain for ROR1, and a binding domain for 4-1BB, A guidance and navigation control (GNC) protein comprising: In the GNC protein, The CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS. the binding domain against PD-L1 comprises VH-CDR1: NTYDMI, VH-CDR2: IITYSGSRYYANWAKG, and VH-CDR3: DYMSGSHL, and VL-CDR1: YSFLAWY, VL-CDR2: SASSLAS, and VL-CDR3: QQGYGKNNVDNA; the binding domain for ROR1 comprises VH-CDR1: GFTISRYHMT, VH-CDR2: HIYVNNDDTDYASSAKG, and VH-CDR3: LDVGGGGAYIGDI, and VL-CDR1: QSSQSVYNNNDLA, VL-CDR2: YASTLAS, and VL-CDR3: AGGYDTDGLDTFA; The binding domain for 4-1BB comprises VH-CDR1: SYHMQ, VH-CDR2: TISSGGNVYYASSARG and VH-CDR3: DSGYSDPM, and VL-CDR1: QASQNIRTYLS, VL-CDR2: AAANLAS and VL-CDR3: QSTYLGTDYVGGA. Guidance and navigation control (GNC) proteins.

6. A guidance and navigation control (GNC) protein as described in claim 5, comprising a chain of amino acid sequences shown in SEQ ID NO: 92 and a chain of amino acid sequences shown in SEQ ID NO:

94.

7. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, CD3 binding domain a binding domain for ROR1, IgG Fc domain, a binding domain for PD-L1, and a binding domain for 4-1BB, A guidance and navigation control (GNC) protein comprising: In the GNC protein, the CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS; the binding domain for ROR1 comprises VH-CDR1: GFTISRYHMT, VH-CDR2: HIYVNNDDTDYASSAKG, and VH-CDR3: LDVGGGGAYIGDI, and VL-CDR1: QSSQSVYNNNDLA, VL-CDR2: YASTLAS, and VL-CDR3: AGGYDTDGLDTFA; the binding domain against PD-L1 comprises VH-CDR1: NTYDMI, VH-CDR2: IITYSGSRYYANWAKG, and VH-CDR3: DYMSGSHL, and VL-CDR1: YSFLAWY, VL-CDR2: SASSLAS, and VL-CDR3: QQGYGKNNVDNA; The binding domain for 4-1BB comprises VH-CDR1: SYHMQ, VH-CDR2: TISSGGNVYYASSARG and VH-CDR3: DSGYSDPM, and VL-CDR1: QASQNIRTYLS, VL-CDR2: AAANLAS and VL-CDR3: QSTYLGTDYVGGA. Guidance and navigation control (GNC) proteins.

8. A guidance and navigation control (GNC) protein as described in claim 7, comprising a chain of amino acid sequences shown in SEQ ID NO: 96 and a chain of amino acid sequences shown in SEQ ID NO:

98.

9. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, CD3 binding domain a binding domain for ROR1, IgG Fc domain, a binding domain for PD-L1, and a binding domain for 4-1BB, A guidance and navigation control (GNC) protein comprising: In the GNC protein, the CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS; the binding domain for ROR1 comprises VH-CDR1: GFTISRYHMT, VH-CDR2: HIYVNNDDTDYASSAKG, and VH-CDR3: LDVGGGGAYIGDI, and VL-CDR1: QSSQSVYNNNDLA, VL-CDR2: YASTLAS, and VL-CDR3: AGGYDTDGLDTFA; the binding domain against PD-L1 comprises VH-CDR1: SGYDMC, VH-CDR2: CIAAGSAGITYDANWAKG, and VH-CDR3: SAFSFDYAMDL, and VL-CDR1: QASQSISSSHLN, VL-CDR2: KASTLAS, and VL-CDR3: CQQGYSWGNVDNV; The binding domain for 4-1BB comprises VH-CDR1: SYHMQ, VH-CDR2: TISSGGNVYYASSARG and VH-CDR3: DSGYSDPM, and VL-CDR1: QASQNIRTYLS, VL-CDR2: AAANLAS and VL-CDR3: QSTYLGTDYVGGA. Guidance and navigation control (GNC) proteins.

10. 10. The guidance and navigation control (GNC) protein of claim 9, comprising a chain of amino acid sequences shown in SEQ ID NO: 100 and a chain of amino acid sequences shown in SEQ ID NO:

102.

11. having an N-terminus and a C-terminus, and in tandem from the N-terminus to the C-terminus, a binding domain for CD19, a binding domain for CD3, IgG Fc domain, a binding domain for PD-L1, and 4-1BB binding domain A guidance and navigation control (GNC) protein comprising: In the GNC protein, the CD19 binding domain comprises VH-CDR1: SSWIG, VH-CDR2: IIYPDDSDTRYSPSFQG and VH-CDR3: HVTMIWGVIIDF, and VL-CDR1: RASQGISSALA, VL-CDR2: DASSLES and VL-CDR3: QQFNSYPFT; the CD3-binding domain comprises VH-CDR1: TNAMS, VH-CDR2: VITGRDITYYASWAKG, and VH-CDR3: DGGSSAITSNN, and VL-CDR1: QASESISSWLA, VL-CDR2: EASKLAS, and VL-CDR3: QGYFYFISRTYVNS; the binding domain against PD-L1, VH-CDR1: SGYDMC, VH-CDR2: CIAAGSAGITYDANWAKG, and VH-CDR3: SAFSFDYAMDL, and VL-CDR1: QASQSISSSHLN, VL-CDR2: KASTLAS, and VL-CDR3: CQQGYSWGNVDNV; The binding domain for 4-1BB comprises VH-CDR1: SYHMQ, VH-CDR2: TISSGGNVYYASSARG and VH-CDR3: DSGYSDPM, and VL-CDR1: QASQNIRTYLS, VL-CDR2: AAANLAS and VL-CDR3: QSTYLGTDYVGGA. Guidance and navigation control (GNC) proteins.

12. A guidance and navigation control (GNC) protein as described in claim 11, comprising a chain of amino acid sequences shown in SEQ ID NO: 108 and a chain of amino acid sequences shown in SEQ ID NO:

110.

13. A nucleic acid encoding the GNC protein according to claim 1 or 2.

14. 14. The nucleic acid of claim 13, having a strand of the nucleic acid sequence set forth in SEQ ID NO: 83 and a strand of the nucleic acid sequence set forth in SEQ ID NO:

85.

15. A nucleic acid encoding the GNC protein according to any one of claims 3 to 10.

16. 16. The nucleic acid of claim 15, having a strand of the nucleic acid sequence shown in SEQ ID NO: 49 and a strand of the nucleic acid sequence shown in SEQ ID NO:

51.

17. A nucleic acid described in claim 15, having a strand of the nucleic acid sequence shown in SEQ ID NO: 91 and a strand of the nucleic acid sequence shown in SEQ ID NO:

93.

18. A nucleic acid described in claim 15, having a strand of the nucleic acid sequence shown in SEQ ID NO: 95 and a strand of the nucleic acid sequence shown in SEQ ID NO:

97.

19. A nucleic acid described in claim 15, having a strand of the nucleic acid sequence shown in SEQ ID NO: 99 and a strand of the nucleic acid sequence shown in SEQ ID NO:

101.

20. A nucleic acid encoding the GNC protein according to claim 11 or 12.

21. 21. The nucleic acid of claim 20, having a strand of the nucleic acid sequence set forth in SEQ ID NO: 107 and a strand of the nucleic acid sequence set forth in SEQ ID NO:

109.

22. CD3, 4-1BB, and The GNC protein according to any one of claims 1 to 12. A cytotoxic cell comprising: A cytotoxic cell, wherein the GNC protein is bound to CD3 and / or 4-1BB via interaction with CD3, 4-1BB, or a combination thereof.

23. A cancer cell comprising EGFR VIII and the GNC protein according to claim 1 or 2, A cancer cell, wherein the GNC protein is bound to the cancer cell via interaction with EGFR VIII.

24. A cancer cell comprising ROR1 and a GNC protein according to any one of claims 3 to 10, A cancer cell, wherein the GNC protein is bound to the cancer cell via interaction with ROR1.

25. A cancer cell comprising CD19 and the GNC protein according to claim 11 or 12, A cancer cell, wherein the GNC protein is bound to the cancer cell via interaction with CD19.

26. CD3, 4-1BB, Cancer cells having EGFR VIII, and The GNC protein according to claim 1 or 2 A biological conjugate comprising: wherein the GNC protein binds to CD3 and / or 4-1BB via interaction with CD3, 4-1BB, or a combination thereof, and the GNC protein binds to cancer cells via interaction with EGFR VIII; biological complex.

27. CD3, 4-1BB, Cancer cells having ROR1, and The GNC protein according to any one of claims 3 to 10. A biological conjugate comprising: wherein the GNC protein binds to CD3 and / or 4-1BB through interaction with CD3, 4-1BB, or a combination thereof, and the GNC protein binds to cancer cells through interaction with ROR1; biological complex.

28. CD3, 4-1BB, CD19-bearing cancer cells, and The GNC protein according to claim 11 or 12. A biological conjugate comprising: wherein the GNC protein binds to CD3 and / or 4-1BB through interaction with CD3, 4-1BB, or a combination thereof, and the GNC protein binds to cancer cells through interaction with CD19; biological complex.

29. A GNC protein according to any one of claims 1 to 12, a cytotoxic cell according to claim 22, or a combination thereof; and a pharmaceutically acceptable carrier, A pharmaceutical composition comprising:

30. 30. A pharmaceutical composition comprising an effective amount of the pharmaceutical composition of claim 29 for treating a subject with cancer.

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