GNC t-cell engager and method of making and using thereof

Abstract

The application provides a multi-specific antibody monomer having a light chain (LC) and a heavy chain (HC), each having a N-terminal and a C-terminal, comprising, a first scFv domain having a binding affinity to a tumor-associated antigen (TAA) at the N-terminal of the HC (Position 1, or P1), a Fab domain having a binding affinity to CD3, and a second scFv domain having a binding affinity to PD-L1 at the C-terminal of the LC (Position 5, or P5) or the N-terminal of the LC (Position 6, or P6). In some embodiments, the TAA comprises EGFR, HER3, DLL3, HER2, GPC3, HLA-G, uPAR, Nectin4, Fra, Tissue Factor, B7-H4, VEGFR2, B7-H3, Muc16, CEACAM6, Claudin6, CLDN18.2, LGR5, GCC, CD20, CD19, Integrin β6, Muc1, CDH6, FAP, CAIX, Integrin β4, CDH17, Lewis B / Y, GPRC5D, CEACAM5, ROR1, or EGFR vIII.

Description

GNC T-CELL ENGAGER AND METHOD OF MAKING AND USING THEREOFCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 63 / 682,278 filed August 12, 2024, under 35 U.S.C. 119(e), the entire disclosure of which is incorporated by reference herein.TECHNICAL FIELDThe application generally relates to the technical field of multi specific antibodies for cancer immunotherapy and, more specifically, to making and using Guidance and Navigation Control (GNC) T-cell engagers having specific structural domain configurations to enhance T cell polyfunctionality.BACKGROUNDImmunotherapy is a vital component of therapy for treating various diseases and conditions such as cancer, autoimmune and infectious diseases, and inflammatory conditions. The cytotoxic nature makes T cells valuable fighters in immunotherapy, especially in cancer immunotherapy. A variety of strategies are devised to make use of T cells for treating cancers, collectively known as cancer immunotherapy, including but not limited to, therapeutic antibody therapy, cell therapy, radioimmunotherapy, and cancer vaccines. Cancer immunotherapy exploits the fact that cancer cells often express tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) on their surface that can be recognized by an antibody or its binding fragment thereof. The other fact is that the design and production of genetically engineered recombinant antibodies has made it possible to produce with monospecific or multispecific monoclonal antibodies. Such an antibody or its binding fragment thereof may be further modified and optimized to produce a therapeutic antibody that can recognize and bind to one or more normal or aberrantly expressed receptors, such as T cell receptor (TCR) complexes, thereby triggering an immune system response. Therapeutic monoclonal antibodies were first commercialized in 1986, followed by antibody-drug conjugates (ADCs) in 2001 and bispecific T-cell engagers (BiTE) in 2014. Chimeric antigen receptor (CAR) T cell therapy, which utilizes T cells transfected with CARs to treat cancer. The first CAR-T therapy was approved in 2017. T cell engagers are highly potent, including cytotoxicity at picomolar doses and observing robust T cell activation. In 2014, the U.S. Food and Drug Administration (FDA) approved Blinatumomab (Amgen, BLINCYTO®), the first anti- CD3xCD19 BiTE for the treatment of acute lymphoblastic leukemia. Subsequent studies have demonstrated that T-cell engagers induced T-cell activation through upregulation of CD25 and CD69, as well as secretion of cytokines (e g., IL-2, TNF-α and IFN-y). Tebentafusp (Immunocore, KIMMTRAK®) is a gplOO peptide-HLA TCR / anti-CD3 fusion protein (as to an antibody) approved for the treatment of metastatic uveal melanoma in 2022(https: / / www.fda.gov / drugs / spotlight-cder-science / bispecific-antibodies-area-research-and- clinical-applications).Bispecific and multispecific antibodies are recombinant antibodies (or their binding fragments) consisting of two and more distinct binding domains capable of binding two and moredifferent antigens or different epitopes of the same antigen (Brinkmann and Kontermann 2017; Kontermann 2012). Bispecific and multispecific antibodies can target multiple disease-modifying molecules as a single drug and with significant advantages over combination therapy or antibody mixtures. In 2021, the FDA issued a guideline for categorizing bispecific antibodies: the bispecific antibodies whose function does not involve bridging two target cells, such as bispecific antibodies targeting two soluble cytokines, binding to different epitopes of the same tumor cell receptor or viral antigen or binding two different targets to mimic the function of endogenous proteins. In this category, bispecific antibody sometimes, but not always, need to bind both targets to be effective. In another category, bispecific antibodies function by connecting two target cells (e g., bispecific antibodies are designed to bring immune effector cells into close contact with a particular TAA, thereby promoting cell killing). Such bispecific antibodies need to bind both targets to be effective. By delivering effectors or effector cells to specific target cells, immune cells have the potential to be re-directed or synergistically effected through the binding of multiple targets, which gives bispecific and multispecific antibodies the ability to guide and navigate effector cells to fight against and kill the target cells, i.e., to activate cytotoxic T-cells to eradicate target cancer cells (Suresh et al. 2014; Kontermann 2012).BiTEs catalyze T cell activation only when they are tethered onto tumor cells using their TAA-binding arm. The tumor cells coated with a certain density of BiTEs facilitate multivalent engagement of the TCR / CD3 complex and activates the T cells through clustering TCR, bypassing the need of MHC-restricted activation of the T cells. The optimized design of BiTEs renders the CD3 agonistic activity only in the tumor site, improving the antitumor efficacy and reducing the side effects of a systemic agonistic T cell activation. As more BiTE products are being developed and trialed in patients, over activated T cells manifest cytokine release syndrome (CRS) and premature T cell exhaustion may occur after a prolonged exposure.PD-L1 (programmed death-ligand 1) and its receptor, PD1 (programmed death- 1), are well known for their role in the induction and maintenance of immune tolerance within the tumor microenvironment. As the tumor progresses, some tumor cells evolve to express PD-L1 as an adaptive immune mechanism to escape anti-tumor responses. Simply, the binding of PD-L1 to PD-1 prevents T cells from killing tumor cells, whereas blocking the binding of PD-L1 to PD-1 with an immune checkpoint inhibitor, such as anti-PD-Ll or anti-PD-1 antibodies, permits the T cells to kill tumor cells. However, not all tumor cells express PD-L1, only 15-30% of patients with various forms of cancer may benefit from the use of therapeutic antibodies against PD1 or PD-L1. On the other hand, 4-1BB is an inducible costimulatory molecule primarily expressed on the surface of activated cytotoxic CD8+ T cells and helper CD4+ T cells. Anti-4-lBB agonistic antibodies solicitate T-cell response to antigens. Therapeutic antibodies targeting 4- IBB have the issue of agonism related to epitope specificity, binding affinity, and off-target effects, leading to a high incidence of immune-related adverse events, such as CRS, immune-mediated colitis, liver toxicity, and dermatologic reactions. A bispecific antibody directed against 4-1BB and PD-L1 exerts anti-tumor effects in a mouse model in which limiting 4-lBB-mediated co- stimulation in tumors contributed to reduced liver toxicity (https: / / www.cancer.gov / research / participate / clinical-trials / intervention / anti-pd-ll-anti-4-lbb-bispecific-antibody-abl503?redirect=true). Similarly, a bispecific antibody directed against CD3 and PD-LlxCD3 can block the PD1 / PD-L1 immune checkpoint and redirect T cells to kill breast cancer cells expressing PD-L1 (https: / / www.cancer.gov / publications / dictionaries / cancer-drug / def / anti-pd-l-anti-cd3-bispecific- antibody-ono-4685).Guidance and Navigation Control (GNC) antibodies are a group of tetraspecific, pentaspecific, and hexaspecific antibody-like proteins characterized by having binding specificities that promote T cell proliferation (via CD3), synergistically stimulate activated T cells (via 4-1BB), and block PD1 / PD-L1 -dependent immune checkpoint signaling. GNC antibodies work by increasing the quality of T cells in the patient before directing them to interact with cancer cells (W02019005641A1, WO2019191120A1, and WO2021092266 Al, incorporated herein in their entirety). Besides the binding of CD3 that engages the proliferation of T cells, the binding of 4-1BB and PD1 / PD-L1 independently promotes the overall function of a GNC antibody. The antagonistic and agonistic effects of binding both PD-L1 and 4-1BB contribute to the overall effectiveness of GNC antibodies against tumor immune responses. In terms of cytolytic activity, GNC antibodies show cytotoxicity at sub-picomolar doses and exhibit advantages over reduced types of bispecific antibodies. These earlier findings suggest that GNC antibodies not only act as T-cell engagers but also exert multimodal activities depending on their composition and cellular environment. As the multiplicity increases, each tetraspecific, pentaspecific, or hexaspecific GNC antibody affords to have at least two binding arms for binding a T cell and a tumor cell, respectively.To differentiate the difference from BiTEs, these GNC antibodies are known as GNC T-cell engagers. In further characterization of complex structural and functional relationships, challenges have emerged in optimization of therapeutic GNC T cell engagers and therapeutic composition of T cells.SUMMARYThe following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. The application relates to a therapeutic agent format capable of enhancing T cell polyfunctionality and improving the efficacy of cancer immunotherapies. Guidance and Navigation Control (GNC) T-cell engagers exhibit multimodal activities depending on their composition and the cellular environment. The structure and binding specificity of this type of T- cell engagers are optimized for re-directing cytotoxic T cells to target and eliminate TAA- expressing cancer cells. Specifically, the application discloses a unifying feature of robust T cell polyfunctionality when the PD-L1 binding domain is allocated to the C-terminus of light chain, also known as position 5. This uniformed structural -functional feature is irrespective of TAA binding domain and present in the absence of TAA antigen binding as demonstrated in both cell- and animal-based tumor models.In one aspect, the application provides multi -specific antibody monomers. In one embodiment, the multi-specific antibody monomer has a light chain (LC) and a heavy chain (HC) each having a N-terminal and a C-terminal. In one embodiment, the monomer comprises a first scFv domain having a binding affinity to a tumor-associated antigen (TAA) at the N-terminal of the HC (Position 1, or Pl), a Fab domain having a binding affinity to CD3, and a second scFv domain having a binding affinity to PD-L. The second scFv domain may be at the C-terminal of the LC (Position 5, or P5) or the N-terminal of the LC (Position 6, or P6).In one embodiment, the TAA comprises EGFR, HER3, DLL3, HER2, GPC3, HLA-G, uPAR, Nectin4, Fra, Tissue Factor, B7-H4, VEGFR2, B7-H3, Mucl6, CEACAM6, Claudin6, CLDN18.2, LGR5, GCC, CD20, CD19, Integrin 6, Mucl, CDH6 , FAP, CAIX, Integrin 04, CDH17, Lewis B / Y, GPRC5D, CEACAM5, R0R1, or EGFR vIII. In one embodiment, TAA comprises EGFR, HER3, DLL3, HER2, GPC3, HLA-G, or uPAR.In one embodiment, the Fab domain comprises complementarity-determining regions (CDRs) comprising: VL CDR1, VL CDR2, and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 109, 110, and 111, and VH CDR1,VH CDR2, and VH having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 112, 113, and 114.In one embodiment, the second scFv domain comprises the CDRs comprising: VL CDR1, VL CDR2, and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 115, 116, and 117, and VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 118, 119, and 120.In one embodiment, the TAA comprises 41BB. In one embodiment, the first scFv domain comprises CDRs comprising: VL CDR1, VL CDR2, and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 121, 122, and 123, and VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 124, 125, and 126.In one embodiment, the TAA comprises uPAR. In one embodiment, the first scFv domain comprises CDRs comprising: VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 127, 128, and 129.In one embodiment, the TAA comprises HLA-G. In one embodiment, the first scFv domain comprises CDRs comprising: VL CDR1, VL CDR2, and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 130, 131, and 132, and VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 133, 134 and 135.In one embodiment, the TAA comprises HER2. In one embodiment, the first scFv domain comprises CDRs comprising: VL CDR1, VL CDR2, and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 136, 137, and 138, and VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 139, 140, and 141.In one embodiment, the TAA comprises GPC3. In one embodiment, the first scFv domain comprises CDRs comprising: VL CDR1, VL CDR2 and VL CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 142, 143, and 144, and VH CDR1, VH CDR2, and VH CDR3 having at least 98%, 99% or 100% sequence identity to SEQ ID NO: 145, 146, and 147.The antibody monomer may tri-specific, tetra-specific, penta-specific, or hexa-specific.The antibody monomer may be tri-specific. In one embodiment, the TAA comprises DLL3, EGFR, or HER2. In one embodiment, the multi-specific antibody monomer has a binding affinity to CD3, PD-L1, and DLL3. In one embodiment, the tri-specific antibody monomer comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 59, 55, or both. In embodiment, the penta-specific antibody monomer comprises CDRs from SEQ ID NO: 59. In embodiment, the penta-specific antibody monomer comprises CDRs from SEQ ID NO:55.The antibody monomer may be tetra-specific. In one embodiment, the monomer may further comprise a third scFv domain at the C-terminal of HC (Position 3, P3). In one embodiment, the third scFv domain has a binding affinity to PD-L1, HER3, or 4-1BB. In one embodiment, the multi-specific antibody monomer has a binding affinity to CD3, PD-L1, a TAA selected from EGFR, DLL3, HER2, GPC3, HLA-G, and uPAR, and one of HER3 and 4-1BBIn one embodiment, the tetra-specific antibody monomer comprises the second scFV domain at P5. In one embodiment, the tetra-specific antibody monomer has the TAA comprising EGFR and the third scFv domain having a binding affinity to HER3. In one embodiment, the tetra- specific antibody monomer has the TAA comprising DLL3 and the third scFv domain having a binding affinity to 4- IBB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising HER2 and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising GPC3 and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising HLA-G and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising uPAR and the third scFv domain having a binding affinity to 4-1BB.In one embodiment, the tetra-specific antibody monomer comprises the second scFV domain is at P6. In one embodiment, the tetra-specific antibody monomer has the TAA c comprising HERZ and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising GPC3 and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising HLA-G and the third scFv domain having a binding affinity to 4-1BB. In one embodiment, the tetra-specific antibody monomer has the TAA comprising uPAR and the third scFv domain having a binding affinity to 4- IBB.In one embodiment, the tetra-specific antibody monomer comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25, 23, 53, 55, 71, 72, 77, 78, 81, 82, 87, 88, 91, 92, 95, 96, 103, 104, 107, or 108. In one embodiment, the tetra-specific antibody monomer comprises a HC having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25, 53, 71, 77, 81, 88, 91, 95, 103, or 107. In one embodiment, the tetra-specific antibody monomer comprises a LC having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23, 55, 72, 78, 82, 88, 92, 96, 104, or 108.In embodiment, the tetra-specific antibody monomer comprises CDRs from SEQ ID NO: 25, 53, 71, 77, 81, 88, 91, 95, 103, or 107. In embodiment, the tetra-specific antibody monomer comprises CDRs from SEQ ID NO: 23, 55, 72, 78, 82, 88, 92, 96, 104, or 108.The antibody monomer may be penta-specific. In one embodiment, the monomer may further comprise a fourth scFv domain attached to the third scFv domain (Position 4 or P4). In one embodiment, the third scFv domain has a binding affinity to PD-L1, HER3, or 4- IBB. In one embodiment, the fourth scFv domain has a binding affinity to 4- IBB orFITC. In one embodiment, the multi-specific antibody monomer has a binding affinity to CD3, PD-L1, EGFR, HER3, and 4- 1BB.In embodiment, the penta-specific antibody monomer may comprise the third scFv domain having a binding affinity to HER3 or PD-L1, and the fourth scFv domain having a binding affinity to 4-1BB.In embodiment, the penta-specific antibody monomer may have the second scFv domain at P5. In embodiment, the penta-specific antibody monomer comprises TAA comprises HER3 or PD-L1.In embodiment, the penta-specific antibody monomer may have the second scFv domain at P6.The third scFv and the fourth scFv may be linked through a linker. In one embodiment, the linker may include 5, 10, 15, 20, 25, 30, or 35 amino acid residues.In embodiment, the penta-specific antibody monomer comprises an amino acid sequence having at least 75%, 805, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21, 23, or both. In embodiment, the penta-specific antibody monomer comprises CDRs from SEQ ID NO: 21 . In embodiment, the penta-specific antibody monomer comprises CDRs from SEQ ID NO: 23.In one embodiment, the monomer may be hexa-specific and further comprises a fifth scFv domain at the P5 or P6. In one embodiment, the second scFv domain is at P5 and the fifth scFv domain is at P6. In one embodiment, the second scFv domain is at P6 and the fifth scFv domain is at P5.In another aspect, the application provides monoclonal antibodies. In one embodiment, the monoclonal antibody comprises the multi-specific antibody monomer as disclosed herein. In one embodiment, the monoclonal antibody is tri-specific, tetra-specific, penta-specific, or hexa- specific.In a further aspect, the application provides isolated nucleic acid sequences encoding the multi-specific antibody monomer, or the monoclonal antibody as closed herein.In a further aspect, the application provides the expression vector comprising the isolated nucleic acid sequence encoding the multi-specific antibody monomer, or the monoclonal antibody as closed herein.In a further aspect, the application provides host cells comprising the isolated nucleic acid sequence encoding the multi-specific antibody monomer, or the monoclonal antibody as closed herein.In a further aspect, the application provides the method for producing the multi-specific monoclonal antibodies. In one embodiment, the method includes the steps of culturing a host cell such that the DNA sequence encoding the monoclonal antibody is expressed, and purifying said monoclonal antibody.In a further aspect, the application provides methods of making the multi-specific monoclonal antibodies. In one embodiment, the method includes the steps of culturing a host cell under conditions wherein said multi-specific monoclonal antibody is produced, and recovering said multi-specific monoclonal antibody.In a further aspect, the application provides immunoconjugates. In one embodiment, the immunoconjugate comprises the multi-specific monoclonal antibody as disclosed herein covalently linked to a cytotoxic agent.In a further aspect, the application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises the multi-specific monoclonal antibodies or the immunoconjugates as disclosed herein, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise a therapeutic agent. In one embodiment, the therapeutic agent cmay be an antibody, an immunoconjugate, a chemotherapeutic agent, a toxin, a radionuclide, or a combination thereof.In a further aspect, the application provides methods for treating or preventing a cancer, an autoimmune disease, or an infectious disease in a subject. In one embodiment, said method may include the step of administering to the subject an effective amount of the multi-specific monoclonal antibodies or the immunoconjugates as disclosed herein.In one embodiment, the cancer may comprise cancer cells expressing EGFR, HER3, DLL3, HER2, GPC3, HLA-G, uPAR, Nectin4, Fra, Tissue Factor, B7-H4, VEGFR2, B7-H3, Mucl6, CEACAM6, Claudin6, CLDN18.2, LGR5, GCC, CD20, CD19, Integrin 06, Mucl, CDH6 , FAP, CAIX, Integrin 04, CDH17, Lewis B / Y, GPRC5D, CEACAM5, R0R1, or EGFR vIII. In one embodiment, the cancer may comprise cancer cells expressing EGFR, HER3, DLL3, HER2, GPC3, HLA-G, uPAR, or a combination thereof.In one embodiment, cancer cell may include a lung cancer cell, a liver cancer cell, a breast cancer cell, a colorectal cancer cell, an anal cancer cell, a pancreatic cancer cell, a gallbladder cancer cell, a bile duct cancer cell, a head and neck cancer cell, a nasopharyngeal cancer cell, a skin cancer cell, a melanoma cell, an ovarian cancer cell, a prostate cancer cell, a urethral cancer cell, a lung cancer cell, a non-small lung cell cancer cell, a small cell lung cancer cell, a brain tumour cell, a glioma cell, a neuroblastoma cell, an esophageal cancer cell, a gastric cancer cell, a liver cancer cell, a kidney cancer cell, a bladder cancer cell, a cervical cancer cell, an endometrial cancer cell, a thyroid cancer cell, an eye cancer cell, a sarcoma cell, a bone cancer cell, a leukemia cell, a myeloma cell, a lymphoma cell, or a combination thereof.The cancer may be a solid tumor. In one embodiment, the cancer is breast cancer, head and neck cancer, head / neck squamous cell cancer, lung adenocarcinoma, squamous cell lung cancer, non-small lung cell cancer, small cell lung cancer, head / neck squamous cell cancer, esophageal cancer, nasopharyngeal cancer, thyroid cancer, kidney cancer, gastric cancer, liver cancer, rectalcancer, colorectal cancer, squamous cell lung cancer, thyroid cancer, bladder cancer, melanoma, glioma, cervical cancer, prostate cancer, breast cancer, uterine / endometrial cancer, pancreatic cancer, ovarian cancer, brain cancer, or papillary kidney cancerIn one embodiment, the cancer comprises lymphoma, leukaemia, or myeloma.In one embodiment, the subject is a human.In one embodiment, the method may include the monoclonal antibody being administered intrathecally, intraperitoneally, or by convection-enhanced delivery.In one embodiment, the method may further comprise co-administering a therapeutic agent, wherein the therapeutic agent comprises an antibody, an immunoconjugate, a chemotherapeutic agent, a toxin, a radionuclide, or a combination thereof.In one embodiment, the therapeutic agent may be an antibody, a chemotherapy agent, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent may include, for example, capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab- palitaxel, a derivative or a combination thereof.The therapeutic agent may be any cancer treating agent or combinations of such agents. In one embodiment, the therapeutic agent may be a tyrosine kinase inhibitor (TKI), an alkylating agent, an anti-metabolite, an anti -microtubule agent, a cytotoxic antibiotic, a topoisomerase inhibitor, a chemoprotectant, or a combination thereof. In one embodiment, the therapeutic agent may be osimertinib, paclitaxel, Docetaxel, Irinotecan, carboplatin, pemetrexed, cisplatin, or a combination thereof.In one embodiment, the tyrosine kinase inhibitor (TKI) comprises Erlotinib, Gefitinib, Icotinib, AZD3759, Sapatinib, Afatinib, Dacomitinib, Deratinib, Poziotinib, Tarlox-TKI, Osimertinib, Nazartinib, Olmutinib, Rociletinib, Naquotinib, Lazertinib, EAI045, CLN081, AZ5104, Mobocertinib, its derivative or a combination thereof.In a further aspect, the application further provides a kit comprising a first container, a second container and a package insert. The first container comprises at least one dose of a first therapeutic composition comprising a multi-specific monoclonal antibody or an immunoconjugate as disclosed herein, the second container comprises at least one dose of a second therapeutic composition comprising a therapeutic agent, and the package insert comprises instructions for treating a subject for cancer using the first and the second therapeutic compositions.In a further aspect, the application provides the solution. In one embodiment, the solution comprises an effective concentration of the multi-specific monoclonal antibody. In one embodiment, the solution is blood plasma in a subject.BRIEF DESCRIPTION OF THE DRAWINGSThe foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged inaccordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:FIGURE 1 shows the P1-P6 positions of antigen binding domains for multi-specific antibodies (1A) and the antibody structures for anti-EGFRxHER3 pentaGNC antibodies (IB) and anti-DLL3 tetraspecific, trispecific, and bispecific antibodies (1C);FIGURE 2 shows redirected T cell cytotoxicity (RTCC) of 3 anti-EGFRxHER3 pentaGNC antibodies of different configurations (SI-77P16, SI-77P17, and SI-77P18) targeting various human cancer cells, including A431 (vulvar squamous cell carcinoma, 2A), A549 (lung cancer, 2B), BxPC-3 (pancreatic adenocarcinomas, 2C); Fadu (hypopharynx squamous cell carcinoma, 2D); KATO III (gastric carcinoma, 2E); LS174T (colorectal adenocarcinoma, 2F); NCI-H727 (colorectal adenocarcinoma, 2G) and UMUC-3 vIII (bladder cell carcinoma, 2H);FIGURE 3 shows the differential cytokine release and polyfunctional responses in CD4 T cells (3A) and CD8 T cells (3B) mediated by 3 anti-EGFRxHER3 pentaGNC antibodies of different configurations (SI-77P16, SI-77P17, and SI-77P18);FIGURE 4 shows the differential cytokine release and polyfunctional responses in CD4 T cells (4A) and CD8 T cells (4B) mediated by 6 anti-DLL3 tetraGNC antibodies of different configurations (SI-83E8, SI-83E15, SI-124X1, SI-124X2, SI-124X3, and SI-124X4);FIGURE 5 shows the differential cytokine release and polyfunctional response in CD4 T cells (5A) and CD8 T cells (5B) mediated by 4 anti-DLL3 trispecific and bispecific antibodies in the presence or absence of anti-PD-Ll domain at the position 5 (SI-124T1, SI-124T2, SI-124X1, and SI- 124X2);FIGURE 6 shows the differential cytokine release and polyfunctional response in CD4 T cells (6A) and CD8 T cells (6B) mediated by 4 anti-DLL3 trispecific antibodies in the presence or absence of anti-PD-Ll domain at the position 5 or anti-4-lBB domain at the position 4 (SI-124T1, SI-124T2, SI-124T3, and SI-124T4);FIGURE 7 shows the positional effect of anti-CD3 domain (αCD3) at Pl position in the absence (as in SI-83X1 and SI-124T3) or presence of anti-PD-Ll domain (aPD-Ll, as in SI-124T1, SL 124E1, SI-83E8) on the internalization under internalizing or non-internalizing conditions after 30 minutes on resting T cells (7A); and the positional effect of anti-CD3 domain (αCD3) atP2 position in the absence (as in SI-124X2 and SL124T4) or presence of anti-PD-Ll domain (aPD-Ll, as in SL124E2, SI-83E15, and SL124T2) on the internalization under internalizing or non-internalizing conditions after 30 minutes on resting T cells (7B);FIGURE 8 shows the effect of GNC T-cell engagers on tumor suppression in xenograft model mice using SI-77P26 (P5-αPD-Ll, pentaGNC antibody) and SI-77P33 (a control antibody with P5-αFITC) (8A); SI-77T5 (P5-αPD-Ll, triGNC T-cell engager) and SI-77x2 (a bispecific T-cell engager) (8B); SL77P26 (P5-αPD-Ll, pentaGNC antibody) and SI-77P32 (a P4-α4-lBB inactive control antibody) (8C); SI-77P26 (P5-αPD-Ll, pentaGNC antibody) and SI-77E14 (P4-α4-lBB- null tetraGNC control antibody) (8D); SI-77P37 (P5-αPD-Ll, P2-αCD3, pentaGNC antibody) and SI-77E13 (P5-αPD-Ll, P2-αCD3, P4-α4-lBB-null tetraGNC control antibody) (8E);FIGURE 9 shows the structures of multi-specific antibodies targeting HER2, HLA-G, GPC3, and uPAR; andFIGURE 10 shows levels of IFNy (10A), Granzyme B (10B), and polyfunctional T cells producing fFNy, IL-2 and TNFa (10C) by GNC T cell engagers with CD3 in P2 position, indicating the positional effect of CD3 and PD-L1 binding on cytokine production (see Table 5).DETAILED DESCRIPTIONIn the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 explicitly contemplated herein.The application discloses, among others, isolated antibodies, methods of making such antibodies, bispecific or multispecific antibodies, pharmaceutical compositions containing the antibodies, bispecific or multispecific antibodies, the methods for making the molecules and compositions, and the methods for treating cancer using the antibodies and compositions disclosed herein.The application relates to methods of making and using multispecific GNC antibodies, in particular, triGNC, tetraGNC and pentaGNC antibodies. The application discloses a class of GNC antibodies characterized by having a PD-L1 binding domain at the position 5 of the light chain and a CD3 binding domain at either position 1 or 2 of the heavy chain (Table 1). The application discloses unexpected T cell polyfunctional response when testing this class of GNC antibodies using both in vitro and in vivo tumor models with a significant implication in cancer immunotherapy.In general, GNC antibodies have a core structure of two immunoglobulin heavy chains (HC), two light chains (LC), with multiple bivalent scFv binding domains covalently linked to the N- and C-terminus of the HC and LC. In the case of hexaGNC antibodies, each heavy chain monomer has 4 positions (from N- to C-terminus, P1-P4) for having an antigen binding domain (D), and each light chain monomer has 3 positions (from N- to C-terminus, P6, P2, and P5). In a typical GNC antibody configuration, DI, D3, and D4 are scFv domains at Pl, P3, and P4 positions, and D6 and D5 are also scFv domains at P6 and P5 positions, respectively. D2 is a Fab region consist of D2-VH and D2-VL at P2-HC and P2-LC on the heavy and light chain monomers, respectively. The name, location, and order of each binding domain and its position are universally applicable for pentaspecific, tetraspecific, trispecific, and bispecific antibodies throughout this application. For example, a PD-L1 binding domain linked to the C-terminus of light chain is expressed as P5-αPD-Ll.GNC antibodies are characterized by having two moieties: moiety 1 comprises, without limitation, binding specificities toward CD3, PD1 / PD-L1, and 4-1BB for GNC antibodies to engage and optimize T cell activation, proliferation, and cytolytic activity; and moiety 2 comprises, without limitation, binding specificities toward one or more TSAs or TAAs on tumor cells. As compared to therapeutic bispecific T-cell engagers, GNC antibodies retain multiple antigen binding domains for engaging immune cells, characterized by having a CD3 binding domain for T cell activation, a 4- IBB binding domain for co-stimulation of T cells, and a PD-L1 binding domain for inhibiting immune checkpoint. In this context, GNC antibodies may be classified as GNC T-cell engagers for the comparative advantage of improved therapeutic efficacy. GNC T-cell engagers contain an Fc domain that allows for FcRn-mediated recycling and half-life extension, as well as facile protein A-based purification. In one embodiment, the Fc domains may be engineered to contain complementary mutations, also known as “knobs-into-holes”, to enhance the formation and stabilization of the heterodimer. Fc receptor-mediated immunity may be incorporated if desired. GNC T-cell engagers are usually larger than an IgG antibody due to increased number of antigen binding domains (AgBD), which provides spatial flexibility for simultaneously binding to both a T cell and a tumor cell. GNC T-cell engagers may be efficacious therapeutics for cancer treatment by targeting one or more tumor antigens, including but not limited to, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, R0R1, CEA, HER2, HER3, EGFR, EGFRvIII, DLL3, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2.In comparison to conventional combination of antibody therapies, multiplicity of binding specificities affords GNC antibodies as a single drug, which not only improves efficacy but also reduces manufacturing cost. Such treatment simplifies clinical administrative standard operation procedure, eases logistical concerns surrounding multi variate dosing, and becomes more affordable to patients.The terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with poly epitopic specificity, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, scFv, bispecific or bi-effective, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens 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 antibodies and fragments mentioned above. In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically bind an antigen. The immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclasses of immunoglobulin molecule. In one embodiment, the antibody may be whole antibodies and anyantigen-binding fragment derived from the whole antibodies. A typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is comprised 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 domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus. Within the variable regions of the light and heavy chains there are binding regions that interacts with the antigen.The term “monoclonal antibody” as used herein 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 naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which 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, the 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 present disclosure 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).Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with 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 chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).Monoclonal antibodies can be produced using various methods including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9: 15-22 (2002) for a review) or from molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453- 7 (2011)). In the present disclosure some antibodies were created by the immunization of rabbits with both human PD-L1 protein and cells transiently expressing human PD-L1 on the cell surface. Rabbits are known to create antibodies of high affinity, diversity and specificity (Weber et al. Exp. Mol. Med. 49:e305). B cells from immunized animals were cultured in vitro and screened toproduce anti-PD-Ll antibodies. Besides immunization of rabbits followed by B cell culture, other common strategies for antibody generation and discovery include immunization of other animals (e.g., mice) followed by hybridoma and / or display on phage, yeast, or mammalian cells; or display using synthetic variable gene libraries. The antibody variable genes were isolated using recombinant DNA techniques and the resulting antibodies were expressed recombinantly and further screened for desired features such as ability to inhibit the binding of PD-L1 to PD-1, 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 like that described in Seeber et al. PLOS One. 9:e86184 (2014). The term “antigen- or epitope-binding portion or fragment” refers to fragments of an antibody that are capable of binding to an antigen. These fragments may be capable of the antigenbinding function and additional functions of the intact antibody. 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 in a single polypeptide chain by a synthetic linker or a Fab fragment which is a monovalent fragment consisting of VL, constant light (CL), VH, and constant heavy 1 (CHI). Antibody fragments are produced using conventional methods known to those skilled in the art. The antibody fragments can be screened for utility using the same techniques employed with intact antibodies.The “antigen- or epitope-binding fragments” can be derived from an antibody of the application by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121 :663-69, Academic Press, 1986.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 yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen. The Fab fragment may contain the constant domain of the light chain and the first constant domain (CHI) 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 CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.“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. It is in this configuration that the three CDRs of each variabledomain 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.The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (k), based on the amino acid sequences of their constant domains.Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, delta, epsilon, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.A “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain “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)).The terms “polypeptide”, “peptide”, and “protein”, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.The term “isolated” means a biological molecule free from at least some of the components with which it naturally occurs. "Isolated," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody," refers to an antibody which is substantially free of other antibodies having different antigenic a binding specificity."Recombinant" means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells.The term “antigen” refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.Also, as used herein, the term “immunogenic” refers to substances which elicit or enhance the production of antibodies, T-cells or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present disclosure to moderate or alleviate the disorder to be treated.The terms "specific binding", "specifically binds to", and "specific for" a particular antigen or an epitope, as used herein, are interchangeable and are defined to mean binding that ismeasurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.The term “affinity” refers to a measure of the attraction between two polypeptides, such as antibody / antigen, receptor / ligand, etc. The intrinsic attraction between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a particular interaction. AKD binding affinity constant can be measured, e g., by Bio-Layer Interferometry, where KD is the ratio of kdis (the dissociation rate constant) to kon (the association rate constant), as KD = kdis / kon.Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10‘4M, at least about 10’5M, at least about IO-6M, at least about IO-7M, at least about IO-8M, at least about IO-9M, alternatively at least about IO’10M, at least about 10'11M, at least about 10'12M, or greater, where KD refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000- , 10,000- or more times greater for a control molecule relative to the antigen or epitope. Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.“Homology” between two sequences is determined by sequence identity. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs. The deviations appearing in the comparison between a given sequence and the above-described sequences of the disclosure may be caused for instance by addition, deletion, substitution, insertion or recombination.“Naive T cells” are thymus-derived mature circulating T cells that have not yet encountered their antigens. A naive T cell must recognize a foreign peptide bound to a self MHC molecule. “Resting T cells” are defined as non-activated T cells that may have recently encountered their specific antigen, are a highly desirable starting material for producing genetically modified CAR- T cells. Priming of naive T cells requires dendritic cell antigen presentation. “GNC-primed T cells” refers to GNC antibody -bounded T cells, such as GNC antibody -bounded naive T cells or resting T cells.The term “cytokine T cell polyfunctionality” refers to the capacity of T cells to produce multiple types of cytokines simultaneously, which is highly advantageous for cancer immunotherapy due to several critical factors. Poly functional T cells can produce a spectrum of cytokines such as interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-α), andinterleukin-2 (IL-2), which are essential for directing and maintaining cytotoxic action against tumor cells. The secretion of diverse cytokines by polyfunctional T cells modulates the tumor microenvironment, rendering it less supportive of tumor growth and more conducive to immune cell infiltration and activity. Cytokines such as IL-2 promote T cell proliferation and survival through autocrine and paracrine signaling, ensuring a sustained immune response by maintaining a robust population of active T cells. Polyfunctional T cells exhibit greater resistance to exhaustion, a state in which T cells lose their effector functions due to chronic antigen exposure, which is prevalent in cancer. The diverse cytokine profile produced by polyfunctional T cells can recruit and activate various immune cells, including natural killer (NK) cells, macrophages, and dendritic cells, thereby creating a coordinated and multi-pronged immune response against the tumor. The combined actions of different cytokines produce synergistic effects, enhancing the overall efficacy of the immune response beyond what any single cytokine could achieve alone.Tumors are often heterogeneous, comprising cells with varying sensitivities to different immune mechanisms. Poly functional T cells can attack multiple aspects of the tumor simultaneously, increasing the likelihood of effective tumor control. Employing multiple cytokines reduces the likelihood of tumor cells escaping immune surveillance through mutations or adaptations in a single pathway.Cytokines such as IL-2 and IFN-y are crucial for the formation and maintenance of memory T cells, providing long-term immunity and rapid responses to tumor recurrence. The presence of polyfunctional T cells is often correlated with better clinical outcomes in cancer patients undergoing T cell-based immunotherapies, such as adoptive T cell transfer or CAR-T cell therapy. Enhancing T cell polyfunctionality can improve the efficacy of these therapies by ensuring that the immune response is potent, sustained, and adaptable to the evolving tumor landscape.The application may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the application has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.EXAMPLESExample 1. Configurations of GNC T-cell engagersGNC T-cell engagers are multispecific antibodies characterized by having multiple binding specificities for T-cells and cancer cells. In contrast, a bispecific T-cell engager consists of one CD3 -binding domain and one TAA-binding domain. A GNC T-cell engager comprises two identical heavy chains and two identical light chains. Each heavy chain monomer may be configured to have up to 4 binding domains, DI (scFv) at Pl, D2 (HC Fab) at P2, D3 (scFv) at P3, and D4 (scFv) at P4, and each light chain monomer may be configured to have up to 3 binding domains, D6 (scFV) at P6, D2 (LC Fab) at P2, and D5 (scFv) at P5. An exemplary tetraGNC heavy chain monomer may link all four binding domains from Pl to P4, ocTAA, αC D3, ocPD-Ll, and ot4-lBB, of which αCD3 and α4-1BB bind to T cells and otTAA and oPD-Ll bind to cancer cells, while the light chain monomer does not require modification. To target EGFR and HER3, the light chain monomer needs to accommodate one binding domain, e.g., at the P5 position. Inthis context, 3 pentaGNC antibodies, SI-77P25, SI-77P26 and SI-77P27 (Figure 1A and Table 1), were generated (see the detailed methods in Example 2) to evaluate the positional effect of αCD3, α4-1BB, and aPD-Ll on RTCC, and T cell polyfunctional cytokine profile. Additional anti- EGFRxHER3 tetraspecific, trispecific, and bispecific T-cell engagers were generated to evaluate the efficacy of tumor suppression in human tumor xenograft models.To optimize GNC T-cell engagers, DLL3 was used as an exemplary single TAA for the design and generation of tetraspecific, trispecific, and bispecific T-cell engagers (Figure IB and Table 1). These anti-DLL3 T-cell engagers were characterized for their efficacy on T cell polyfunctional cytokine profile and internalization. The aDLL3 domain was derived from a humanized monoclonal antibody (5018A1) that bind DLL3 with high affinity KD value. Its binding affinity may influence the efficacy and safety profile of a therapeutic antibody due to its primary sequences (e.g., CDRs), secondary structure (e.g. scFv and Fab), and location in a multispecific antibody. For example, when targeting tumors with heterogenous antigen expression, antibody-dependent cellular cytotoxicity (ADCC) directed towards tumors with low density antigen expression is often more efficient with high affinity antibodies. Conversely, high affinity antibodies, particularly those with slow dissociation rates, can lead to unwanted toxicity. 5018A1 binds to DLL3 and has a binning site by bio-layer interferometry indicative of a distinct epitope.Example 2. Generation and characterization of GNC antibodies Codon-optimized coding regions encoding antibody heavy and light chains preceded byKozak and secretory signal peptide sequences were cloned into the pTT5 vector using standard molecular biology techniques. Antibodies were expressed by transiently transfecting the expression plasmids for heavy and light chains in the ExpiCHO system (Thermo Fisher). Briefly, 10 pg of each expression plasmid was brought to 1 m with OptiPRO SFM medium. 1 m of OptiPRO SFM medium containing 80 pL Expifectamine CHO reagent was added to the DNA and incubated at room temperature for 2.5 minutes. The resulting mixture was then added to 25 mb ExpiCHO cells at 6x106 cells / mL in a 125 mb Erlenmeyer flask and incubated at 37°C, 5% CO2, and 150 rpm. Cells were fed with 8.75 mb ExpiCHO feed and 150 pL of CHO enhancer at 24 hours post-transfection and shifted to 32°C, 5% CO2, and 150 rpm. Cells were fed again at 48 hours post-transfection with 8.75 mb ExpiCHO feed. Culture supernatant was harvested 9 days post-transfection, by spinning for 15 minutes at 4,500 rpm to pellet the cells and passing the supernatant through a 0.2 mm filter. Expression titer was quantitated using biolayer interferometry on an Octet384 system with protein A sensors and a standard curve prepared with purified antibody protein.Proteins were purified from the harvested supernatant using a 5-ml MabSelect PrismA protein A column (GE Healthcare) on an Akta Avant FPLC system (GE Healthcare). The column was equilibrated with phosphate-buffered saline (PBS, 125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8). The supernatant was loaded at a flow rate of 1 mL / min onto a phosphate- buffered saline equilibrated column. The column was washed with 10ml PBS prior to eluting thebound protein with 5 mL of 50 mM sodium acetate, pH 3.5. The eluted protein was immediately neutralized by addition of 0.5 mL IM Tris-Cl, pH 8.0.Immediately after protein A purification, proteins were analyzed by analytical SEC using Waters Acquity UPLC H-Class with ACQUITYUPLC® Protein BEH SEC 200 A, 4.6 mm x 150 mm, 1.7 pm column. 10 pg of protein was injected onto the column with a flow rate of 0.3 mL / min in a mobile phase of PBS. Proteins were further purified by preparative SEC using Superdex Increase 10 / 300 GL column in a mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5, ultimately to be buffer-exchanged into 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. Final samples contained >95% protein of interest as assessed by analytical SEC and were used for subsequent assays.Biolayer interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify binding kinetics of bispecific antibodies to EGFR and EGFRvIII. Antibody was captured to anti-human Fc (AHC) sensor tips by loading for 180 seconds at 5 pg / ml. After a 60- second baseline step, a 180-second association phase with serial dilutions (0-100 nM; 1 :2 dilution factor) of His-tagged EGFR or EGFRvIII (expressed and purified in-house) in assay buffer (PBS with 0.1% BSA, 0.05% Tween20) was performed, followed by a 300-second dissociation phase in assay buffer. Regeneration was achieved using 10 mM glycine, pH 1.5. Binding curves were globally fit to a 1 : 1 model to extract the dissociation constants (KD) and kinetic association and dissociation rates.Biolayer interferometry (Octet) was used for epitope binning studies. Biotinylated EGFR (Aero EGR-H82E3) or EGFRvIII (Aero EGR-H82E0) was immobilized at 1 pg / mL for 180 seconds onto streptavidin sensors. After a 30-second baseline in assay buffer, association with 100 nM of the first antibody was performed for 300 seconds. Finally, association with 100 nM of the second antibody was performed for 300 seconds. Octet Analysis 12.0 software was used to generate epitope binning matrices.Example 3, Redirected T cell cytotoxicity (RTCC) of GNC antibodiesRedirected T-cell Cytotoxicity AssayFluorescent reporter-expressing cell lines (A431, BxPC3, Fadu, Kato-III, LS174T, NCI- H727, A-549, UMUC3-vIII) were generated using nuclear Red lentiviral particles from Essen Biosciences via lentiviral transduction. T cells from peripheral blood mononuclear cells (PBMC) from normal donors were enriched from blood acquired from Bloodworks Northwest (Seattle, WA, USA) using the RosetteSep (Catalog # 15061) and isolated through gradient centrifugation with Ficoll Paque PLUS (Cytiva). Cancer cell lines and PBMC derived T cells were stored at -196°C until use. Tumor cells were co-cultured with T cells at ration of 10 CD3+ cell: 1 Target cell in the presence of titrated T cell engagers for 37 hours with the target cell reporter signal detected by time-series fluorescent microscopy using the Incucyte S3. Assay conditions were conducted in ultra-low attachment (ULA) 384-well round-bottom plates. Dose-response curves were modeled using a four-parameter symmetrical fit in Prism 9.31 (GraphPad).To evaluate the potential therapeutic effect of pentaGNC antibodies, eight cancer cell lines were used to measure the RTCC activity mediated by three anti-EGFRxHER3 pentaGNCantibodies, namely SI-77P25, SI-77P26, and SI-77P27, respectively. As depicted in Table 1, each of these antibodies comprises the same set of five binding domains but in different configurations. SI-77P25 and SI-77P27 were configured to have ccCD3 at Pl and P2, respectively, whereas SI- 77P26 was configured to move ocPD-Ll from its fixture position at P4 to P5 on the light chain, as the exemplary anti-EGFRxHER3, P5-αPD-Ll pentaGNC antibody.The EGFR and HER3 expressing cancer cell lines were incubated with purified peripheral resting pan-T cells at a 10: 1 effector to target cell ratio in the presence of GNC antibodies. The number of live cells was monitored over time using a fluorescence microscopy that tracks a reporter protein signal. Results at 37 hours were plotted and data were fit to a non-sigmoidal curve model (Figure 2).A431, a cell model for vulvar squamous cell carcinomaA431 is a squamous cell carcinoma expressing both EGFR and HER3. Similar dosedependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2A), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in A431. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αCD3 was present at the Pl position.A549, a cell model for lung adenocarcinomaA549 is a lung adenocarcinoma expressing both EGFR and HER3. Similar dose-dependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2B), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in A549. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αC D3 was present at the Pl position.BxPC-3, a cell model for pancreatic adenocarcinomasBxPC3 is a pancreatic adenocarcinoma expressing EGFR and HER3. Similar dosedependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2C), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in BxPC3. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αCD3 was present at the Pl position.Fadu, a cell model for head and neck squamous cell carcinomaFaDu is a hypopharyngeal squamous cell carcinoma expressing EGFR and HER3. Similar dose-dependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2D), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in Fadu. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αCD3 was present at the Pl position.KATO III, a cell model for gastric carcinomaKato-III is a gastric adenocarcinoma expressing EGFR and HER3. Similar dose-dependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2E), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in Kato-III. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αC D3 was present at the Pl position.LS I 74T, a cell model for colorectal adenocarcinomaLS174T is a colon adenocarcinoma expressing EGFR and HER3. Similar dose-dependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2F), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in LS 174T. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αCD3 was present at the Pl position.NCI-H727, a cell model for colorectal adenocarcinomaNCI-H727 is a lung cancer line expressing EGFR and HER3. Similar dose-dependent RTCC activities were observed for SI-77P25, SI-77P26 and SI-77P27 (Figure 2G), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in NCI-H727. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when otCD3 was present at the Pl position.UMUC-3 vIII, a cell model for bladder cell carcinomaUM-UC-3-vIII is a urothelial bladder carcinoma expressing 2 forms of EGFR that can both be bound by the GNC aEGFR domain, as well as HER3, making it potentially susceptible to RTCC by the GNC tested. Similar dose-dependent RTCC activities were observed for SI-77P25, SI- 77P26 and SI-77P27 (Figure 2A), indicating that the αCD3 domain at the Pl or P2 position have overlapping RTCC activities in UM-UC-3-vIII. Similarly, aPD-Ll at the P5 or P3 positions (i.e., aHER3 at the P5 or P3 position) did not affect RTCC activity when αCD3 was present at the Pl position.These results indicate that the GNC antibodies can bind directly to TAA and activate pan- T cells. In RTCC analysis, a combination of five binding domains (but not necessarily in the same configuration) in the GNC antibody had similar cytolytic activity against eight different cancer cell lines.Example 4. P5-αPD-L1 unleashes polyfunctional cytokine production in GNC-primed T cellsGNC antibodies are characterized by having CD3, PD-L1, and 4-1BB binding domains for regulating T cell proliferation and activation, while retaining the ability to have additional TAA binding domains. When T cells encounter GNC antibody drugs in the peripheral blood, naive T cells are first primed prior to priming TAA-expressing tumor cells elsewhere in case of solid tumors and soft tissue sarcoma. Such GNC-primed T cells have not been well -characterized, and how GNC-primed T cells recycle or navigate through heterogeneous cellular environments remains unknown. The heterogeneous cellular environment refers to the circulating PBMC and tumor microenvironment where lymphocytes and myeloid cells are permanently present. Therefore, an innovative cell culture system is needed to assess the structure-function relationship of GNC antibodies in the presence of any TAA, such as EGFR, HER3, or DLL3.T cell polyfunctional cytokine production has been used as a valuable biomarker for assessing therapeutic responses in CAR-T therapy, cancer vaccines, T cell engagers, and natural immunity for cancer and viral diseases. However, in each of these situations, this biomarker is used to characterize post-activation T cells. In case of GNC antibodies, naive T cells are augmented with additional monocytes, resulting in sufficient interactions in the peripheryincluding binding of CD3 and 4-1BB on T cells and PD-L1 on monocytes. Optimized GNC antibodies may enhance the quality of GNC-primed T cells prior to encountering tumor cells. In this context, the application employs an assay system, i.e., T-Cell Polyfunctional Cytokine Profiling System, to evaluate GNC-primed T cells. As compared to conventional T-cell cytotoxic or cytolytic analyses that only quantify the level of cytokines, this profiling system profiles cytokines released by individual T cells.T cell polyfunctional cytokine profiling systemTo evaluate T cell polyfunctionality through intracellular cytokine flow cytometry, PBMCs or isolated T cells were co-cultured with monocytes and activated using specific materials, namely SI-77P25, SI-77P26, and SI-77P37. As a positive control, PMA(Phorbol 12-myristate 13-acetate) and ionomycin were used to ensure robust activation. The cells were cultured in X-Vivo-15 medium and incubated for 24 hours. During the last 7 hours of incubation, Brefeldin A and Monensin were added to inhibit protein transport and allow for the accumulation of intracellular cytokines. Following incubation, the cells are harvested and stained for surface markers, including TCRα / β, CD4, and CD8, to identify T cell subsets. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized using a saponin-based buffer, and stained intracellularly with fluorochrome-conjugated antibodies specific for IFN-y, IL-2, TNF-α, and Granzyme-B. The stained cells were then analyzed using a BD Fortessa flow cytometer to assess cytokine production and determine the polyfunctionality of the T cells. Data was collected and analyzed to evaluate the proportion of T cells expressing multiple cytokines, providing insights into their functional capacity.Anti-EGFRxHER3, P5-αPD-Ll pentaGNC antibodiesThree anti-EGFRxHER3, P5-αPD-Ll pentaGNC antibodies, SI-77P25, SI-77P26, and SI- 77P27, were generated and characterized (see Example 1) to evaluate the positional effect of αCD3, -α4-1BB, and ocPD-Ll domains. SI-77P25 and SI-77P27 were configured to have Pl- αCD3 and P2-αC D3, respectively. SI-77P26 was configured to have αPD-Ll from its previously fixed position at the P3 position on the heavy chain to the P5 position on its light chain, resulting a new class of an anti-EGFRxHER3, P5-αPD-Ll pentaGNC antibody.The effects of all three GNC antibodies on T cell polyfunctional cytokine release profiles were evaluated in the absence of TAA-expressing cells. After 48 hours of priming by GNC antibodies, the GNC-primed CD4 and CD8 T cells were fixed, stained and counted to observe the release of polyfunctional cytokines, i.e., the simultaneous expression of IL-2, TNFa, IFNy, and GZB in individual T cells (Figure 3A and 3B). While all GNC antibodies induced dose-dependent release of polyfunctional cytokines in individual CD4 and CD8 T cells, the induction by SI-77P26 was dramatically enhanced, reaching over 30% of CD4 T cells and over 40% of CD8 T cells at the 100 pM of antibody.This finding was unpredictable as all three antibodies comprise the same set of five binding domains and exert comparable levels of cytolytic activity in RTCC assay. SI-77P26 differs from the other antibodies by having aPD-Ll domain at the P5 position. Between SI-77P25 and SI- 77P27, the difference is the location ofαC D3 domain at the Pl and P2 position, respectively. Thefact that the two antibodies induced similar levels of polyfunctional CD8 T cells indicate that the αC D3 position does not affect the release of polyfunctional cytokines by CD8 T cells. In normal PBMC, the ratio of CD4 and CD8 T cells is about 2: 1. When compared the ratio of polyfunctional CD8 T cells over polyfunctional CD4 T cells that were induced by GNC antibody at 1000 pM, SI- 77P25 and SI-77P26 induced T cells at a ratio greater than 1.5. This observation implies that CD8 T cells are more sensitive to GNC-priming. Indeed, SI-77P27 led to a ratio of great than 3, implying disproportional less polyfunctional CD4 T cells.The data reveal a new class of pentaGNC antibody characterized by having P5-otPD-Ll to enhance polyfunctional cytokine release in GNC-primed CD4 T cells and CD8 T cells independent of TA As.Anti-DLL3, P5-αPD-Ll tetraGNC antibodiesPrior to become activated, GNC-primed T cells have increased production of polyfunctional cytokines without GNC antibody binding to TAAs. To recapitulate the P5-αPD- L1 mediated T cell polyfunctional cytokine production, a group of tetraspecific, trispecific, and bi specific T cell engagers was generated and characterized to target DLL3 (Table 1 and Figure IB). DLL3, also known as Delta-Like Ligand 3, is an inhibitory Notch ligand highly expressed in small cell lung cancer (SCLC) and other neuroendocrine tumors but minimally expressed in normal tissues, which makes DLL3 an ideal TAAfor target therapy.The bispecific, trispecific and tetraspecific structural variants allowed the specific testing of domain contributions to T cell polyfunctionality. As in the previous experiments, the PBMC and monocyte co-culture system was employed. Frequency of CD4 and CD8 T cells expressing cytokines TFNy, TNFa, IL-2, GZB and MIP-1β were measured, and T cell polyfunctionality is defined as the simultaneous expression of 3 or more of these cytokines in subpopulations of individual T cells. In the first experiment using this set of molecules, the ability of tetraspecific GNC with variable positional conformations of αC D3, 014-1BB, aPD-Ll and aDLL3 domains to generate polyfunctional T cells was evaluated (Figure 4A and 4B). While all GNCs could generate dose-dependent polyfunctionality in T cells in CD4 and CD8 populations, the induction by SL 83E15 was dramatically enhanced compared to other anti-DLL3 GNC antibodies tested at 100 pM and 1000 pM concentrations, reaching nearly 15% in CD4 and CD8 T cells at the 100 pM dose level. Based on the positions of αC D3, α4-lBB, aPD-Ll domains, the results indicate that, by having P5-otPD-Ll, SI-83E15 and SL124E1 were enabled to enhance the production of polyfunctional cytokines in both CD4 and CD8 T cells. In addition, the ratio of the poly functional CD8 T cells over CD4 T cells induced by either SI-83E15 or SL124E1 was greater than 1, which was consistent with the observation of pentaGNC antibodies targeting EGFR and HER3 (Figure 3 A and 3B). SI-83E15 and SI-124E1 have P5-αPD-Ll paired with P2-αCD3 and P1- αCD3, respectively. SI-83E8 and SI-83E2 have P3-O.PD-L1 paired with Pl-αCD3 and P2- αCD3, respectively. The result shows that a higher level of T cell polyfunctional cytokine production induced by SI-83E15 over SL124E1 (Figure 4A and 4B), suggesting that the pair of P5-αPD-Ll with P2-αCD3 is more efficacious than the pair of P5-αPD-Ll with Pl-αCD3. To furtherinvestigate the positional effect of αC D3, SI-124E3 and SI-124E4 were created to pair P3-αPD- L1 with P5-αCD3 and P2-αCD3, respectively. SI-124E3 and SI-124E4 induced dose-dependent increase of CD8 T cell polyfunctional cytokine production at levels comparable to those induced by SI-83E8 and SI-83E2. When compared to the effect of pentaGNC antibodies, the tetraGNC antibodies also induced T cell polyfunctional cytokine production but at lower levels regardless the ocPD-Ll domain at either P5 or P3 position.Anti-DLL3, P5-αPD-Ll trispecific vs. bispecific T-cell engagersTo assess how P5-αPD-Ll promotes T cell polyfunctional cytokine release, two bispecific T-cell engagers, SI- 124X1 and SI- 124X2, were generated with the αC D3 domains at the Pl and P2 position, respectively. Then, the oPD-Ll domain was placed to the P5 position of the two bi specific T-cell engagers, resulting two tri specific T-cell engagers, SI-124T1 and SI-124T2 (Figures 5A and 5B). While the two bispecific T-cell engagers were unable to induce any significant polyfunctional cytokine release in CD4 and CD8 T cells, both SI-124T1 and SI-124T2 induced robust response in CD4 T cells and CD8 T cells characterized by the ratio of CD8 T cells over CD4 T cells similar to the ratio induced by pentaGNC and tetraGNC antibodies (see Figure 3 and Figure 4). The result indicates that P5-αPD-Ll alone is sufficient to enable a bispecific T- cell engager to stimulate polyfunctional cytokine production in T cells.Anti-DLL3 trispecific T-cell engager having either P5-αPD-L1 or P4-α4-lBBTo assess how α4-1BB alone may affect T cell polyfunctional cytokine release, the α4- 1BB domain was placed to the P3 position of SI-124X1 and SI-124X2, resulting two trispecific T- cell engagers, SI-124T3 and SI-124T4. Together with SI-124T1 and SI-124T2, the two sets of trispecific T cell engagers were evaluated for their ability to generate polyfunctional T cells. The result shows a clear distinction in their ability to induce polyfunctional T cells (Figure 6A and 6B). The trispecific T-cell engagers with α4-lBB (SI-124T3 and SI-124T4) did not induce any significant polyfunctional cytokine production in either CD4 or CD8 T cells, while SI-124T1 and SI-124T2 induced a robust dose-dependent response in polyfunctional T cells.Overall, the effect of P5-αPD-Ll to multispecific T-cell engagers in promoting polyfunctional cytokine production in T cells was surprising. Further analysis revealed several characteristic features. First, the effect of a PD-L1 binder on T cell polyfunctionality was P5 position-specific; GNC-priming had a greater effect on CD8 T cells than on CD4 T cells; and this effect was potentiated by αC D3 at the P2 (Fab) position as compared with the P2 (scFv) position. Example 5, Positional effect of αCD3 and aPD-Ll domains on internalization in resting T cells Antibody binding and internalization in resting T cellsTo determine the extent of antibody internalization in the continuous presence of the antibody drug, resting human T cells are prepared in X-vivo 15 media. The antibody of interest is titrated to generate a dose-response curve. The cells are divided into two groups: one incubated with sodium azide to block internalization and the other without sodium azide. Both groups are incubated with the antibody for 30 minutes at 4°C to prevent internalization. Following incubation, cells are washed with PBS and stained with fluorochrome-conjugated anti-Fc Fabs to detect surface-bound antibodies. The fluorescence intensity is measured using a BD Fortessa flowcytometer. Data analysis involves comparing the area under the curve (AUC) and binding maxima between the internalizing and non-internalizing conditions, with the use of sodium azide serving as a method to determine the extent of differential antibody internalization in the continuous presence of the antibody drug.To interrogate internalization of the GNC antibodies having an 0.CD3 domain in either Pl or P2 position (see Figure 1, Pl-αCD3 GNC or P2-αCD3 GNC, respectively), resting T cells were exposed to GNC antibodies for 30 minutes under internalizing and non-internalizing conditions. Surface quantification demonstrates that the Pl-αCD3 GNC antibodies without aPD-Ll domains did not internalize in resting T cells over 30 minutes (SI-83X1 and SI-124T3, Figure 7), even though the surface abundance of αCD3 molecules increased ranging from 1.3 to 45% in internalizing conditions over non-internalizing conditions. In contrast, the Pl-αCD3 GNC antibodies having an aPD-Ll domain at either P3 (SI-83E8) or P5 (SI-124T1, SI-124E1) position induced rapid internalization. The surface abundance of αCD3 molecules reduced ranging from - 39.1% to -61.3% in internalizing over non-internalizing conditions, indicative of increased activity of internalization in resting T cells due to the addition of an aPD-Ll domain.The P2-αCD3 GNC antibodies induced the internalization in resting T cells in the absence of an aPD-Ll domain (SI-124X2 and SI-124T4, Figure 7A), ranging from -59.4% to -65.2% in internalizing over non-internalizing conditions. However, an addition of an aPD-Ll domain in either P3 (SI-124E2) or P5 (SI-83E15, SI-124T2) did not significantly change the level of internalization, showing similar surface staining ranging from -42% to -78.3% in internalizing over non-internalizing conditions.Taken together these data reveal characteristic differences between the Pl-αCD3 GNC antibodies and the P2-αCD3 GNC antibodies. Pair-wise comparisons of the GNC antibodies in this experiment showed that P2-αCD3, but not Pl-αCD3, induced a basal level of internalization when bispecific antibodies (SI-83X1 vs. SI-124X2) or trispecific antibodies (SI-124T3 vs. SI- 124T4) were used to prime the resting T cells (Figure 7A and Figure 7B). The tetraspecific antibodies, having an aPD-Ll domain at either P3 or P5 position, induced comparable levels of internalization. In contrast, the levels of internalization induced by P2-αCD3 antibodies seem to be similar between bispecific, trispecific, and tetraspecific antibodies (Figure 7A). One simple scenario is the difference in domain structure. Pl-αCD3 is a single-chain variable fragment (scFv), whereas P2-αCD3 is a Fab region consisting of the light chain and heavy chain variable domains. The other scenario is the proximity of αC D3 to P5-αPD-Ll . The data showed that P2- αCD3, but not P2- αCD3, in bispecific and trispecific T-cell engagers led to a background level of internalization in resting T cells, and that the addition of the aPD-Ll domain at the P3 or P5 position positively impact internalization increase. Incidentally, the level of T cell poly functional cytokine production correlates with internalization activity (SI-124E1 and SI-124E2 in Figure 4 and 7). The internalization of resting T cells is indicative of endocytosis usually associated with downregulation of immunological synapse and recycling of the TCR:CD3 complex. The binding of CD3 to crosslink the TCR:CD3 complex may mimic the formation of immunological synapse. In this context, T cells may respond differently to the crosslink mediated by scFv versus Fab.Example 6. P5-αPD-Ll extends GNC antibody-guided tumor suppression in vivoXenograft tumor modelIn this method for assessing tumor growth in mouse xenograft models, the FADU Mixeno Model (DT2023PD114) is used in NOG mice. These mice are immunocompromised, facilitating the engraftment of tumor cells. The FADU Mixeno cells have specific receptor counts: EGFR at 323,390 counts per cell, HER3 at 9,544 counts per cell, and PD-L1 at 38,265 counts per cell. Additionally, the mice are engrafted with human peripheral blood mononuclear cells (PBMCs) to allow evaluation of redirected T cell activity toward FADU. The mice are subcutaneously injected with a known quantity of these tumor cells at a predetermined site, typically in the flank region. After allowing the tumors to establish and reach a measurable size (around 5-10 mm in diameter), the mice are randomly assigned to different treatment groups, ensuring an equal distribution of initial tumor sizes. Treatment administration begins on Day 0, following the designated protocol, which may include chemotherapy, targeted therapy, or immunotherapy. Tumor size is measured using digital or Vernier calipers at the start of the treatment (Day 0) and on Days 3, 7, 11, 14, and 18 during the 21 -day treatment period. For each measurement, the longest diameter (L) and the perpendicular diameter (W) of the tumor are recorded. The mean and standard error of the mean (SEM) for the tumor area are calculated and plotted for each treatment group to evaluate the efficacy of the treatment protocols.To investigate the efficacy of P5-αPD-Ll GNC antibodies in vivo, the following experiments were carried out to evaluate xenograft tumor reduction in an immunodeficient mouse model. Human tumor cells expressing the drug target proteins EGFR, HER3 and PD-L1 were implanted in mice lacking a normal functioning immune system (NOG mice), which provides an in vivo model of human tumor growth. NOG mice were subcutaneously inoculated with tumor cells (1.3 x 10 6 / mouse) and intravenously inoculated with huPBMC (5x 10A6 / mouse) to establish mixeno model on the same day. Mice with tumors are then treated by i.v. injection with human immune cells and GNC antibodies such that the GNC antibodies direct the human immune cells toward killing tumors. Two sets of experiments were carried out comprising 3 experimental groups and 5 mice per group.P5-αPD-Ll pentaGNC antibodies In the first experiment, mice received tumor grafts from the cancer cell line Fadu (derived from a hypopharyngeal tumor), and human peripheral blood mononuclear cells (PBMC), as a source of immune cells. Mice were divided into 3 groups: treated with either SI-77P26 (with P5- ocPD-Ll, at 0.250 mg / kg dose), SI-77P33 (with P5-αFITC, at 0.350 mg / kg to adjust the replacement of ocPD-Ll domain by 4D5, an ocFITC domain at P5 position, to molecular equivalent dose of SI-77P26), or a vehicle control (0.9% sodium chloride). Mice were dosed intravenously with indicated antibodies weekly for a period of 21 days (QW*3). The tumor size in each mouse was measured using calipers at the start of the treatment (Day 0) and again on Days 3, 7, 11, 14 and 18 of the 21 -day treatment period. The mean and standard error of the mean in tumor area was then calculated and plotted for each treatment group and shown in Figure 8A.The results show that the average size of tumors in mice receiving either SI-77P26 or SI- 77P33 was smaller than that in the vehicle control group of mice, with significant differences observed in antibody drug vs vehicle treatment group at Day 3 through Day 18. By Day 18, the average size of tumors increased from Day 0 by a factor of 8.3 -fold in the vehicle treated group, 1.60-fold in the SI-77P26 group, and 4.7-fold in SI-77P33 having P5-αPD-Ll replaced by P5- ocFITC domain (4D5). This data indicated that P5-αPD-Ll improves the efficacy of SI-77P26 in suppressing tumor growth.P5-αPD-L1 trispecific T-cell engagerThe second experiment was to determine whether P5-O.PD-L1 alone is sufficient to improve the efficacy of a bispecific T-cell engager. The same type of xenograft tumor model was used, and the mice received Fadu tumor cell grafts and immune cells of the PBMC origin. Mice were divided into 3 groups and subsequently treated with a (Pl-αEGFR)x(P2-αCD3)x(P5-αPD- Ll) trispecific T-cell engager (SI-77T5, 0.250 mg / kg dose), a (Pl-αEGFR)x(P2-αCD3) bispecific T-cell engager (SI-77X2, but dosed at the molecular equivalent of SI-77T5, 0.200 mg / kg); or a vehicle control (0.9% sodium chloride). Mice are dosed intravenously with drug or vehicle control weekly for a period of 21 days (QW*3). The change in tumor sizes in each mouse was measured using calipers at the start of the treatment (Day 0) and again on Days 3, 7, 11, 14, 18 and 21 of the 21 -day treatment period. The mean and standard error of the mean in tumor area was then calculated and plotted for each treatment group (Figure 8B).The results show that the average size of tumors in the mice receiving either SI-77T5 or SI-77X2 was significantly smaller than that in the mice of vehicle control group, with significant differences observed in antibody drug vs vehicle treatment group at Day 11 through Day 18. By Day 18, the average tumor volume increased from Day 0 by a factor of 16.8-fold in the vehicle treated group, 0.15-fold in SI-77T5, and 4.6-fold in SI-77X2. These results indicate that, while SI-77X2 acted as an exemplary bispecific T cell engager to suppress tumor growth, the addition of P5-otPD-Ll to a bispecific T-cell engager extended the tumor suppression effect. Thus, P5- otPD-Ll independently contributes cytotoxic activity to T-cell engagers and extends the inhibition of tumor growth in vivo.P4-α4-lBB in pentaGNC antibodiesAs the prior art to the application, each of ocPD-Ll and α4-1BB domains at P3 and P4 positions on the heavy chain monomer (i.e. no modification to the light chain monomer) of a tetraGNC antibody improves the efficacy of redirected T cell cytotoxicity. Herein, the positional effect of P5-αPD-Ll was revealed by profiling T cell polyfunctional cytokine release, not by RTCC, and by measuring the tumor suppression effect of a P5-αPD-Ll trispecific T-cell engager in a xenograft tumor model (Figures 2, 3A, 3B, and 8B). To assess the contribution of lBB to the cytotoxic activity of P5-αPD-Ll GNC antibodies, NOG mice received Fadu tumor cell grafts and human PBMC, and were divided into 3 groups to receive: SI-77P26 that carries a P4-α4-lBB (clone 466F3) domain (1.071 mg / kg dose); SI-77P32 that carries a P4-αFITC (clone 4D5) as a control at the molecular equivalent of SI-77P26 (1.5 mg / kg), and a vehicle control (0.9% sodium chloride), respectively. Mice were dosed intravenously with drug or vehicle control weekly for aperiod of 21 days (QW*3). The size of the tumor in each mouse was measured using calipers at the start of the treatment (Day 0) and again on Days 3, 7, 11, 14 and 18 of the 21-day treatment period. The mean and standard error of the mean in tumor area is then calculated and plotted for each treatment group (Figure 8C).The results show that the average size of tumors in mice receiving either SI-77P26 or SI- 77P32 was smaller than that in the vehicle control group of mice, with significant differences observed in drug vs vehicle treatment group at Day 3 through Day 18. By Day 18, average tumor volume increased from Day 0 by a factor of 8.3-fold in the vehicle treated group, 0.06-fold in the SI-77P26 group, and 0.02-fold in the SI-77P32 group that lacks the binding activity from the P4 position. Thus, under this in vivo assay condition, the 4-1BB binding activity, or lack of it, exerts no overt effect to the increased level of tumor suppression that is mediated by this P5-O.PD-L1 pentaGNC antibody.P l -αCD3, P4-α4-lBB-null tetraGNC antibodiesTo confirm any role of P4-α4-lBB to the cytotoxic activity of P5-αPD-Ll GNC antibodies in vivo, a P4-α4- IBB -null tetraGNC antibody, SI-77E14, was generated and characterizaed. NOG mice received Fadu tumor cell grafts and human PBMC, and were divided into 3 groups to receive: SI-77P26, which a pentaGNC antibody having a P4-α4-lBB (clone 466F3) domain (1.071 mg / kg dose); SI-77E14 , which is a tetraGNC antibody without a lBB at P4 pαo4s-ition at the molecular equivalent of SI-77P26 (1.286 mg / kg), and a vehicle control (0.9% sodium chloride), respectively. Mice were dosed intravenously with drug or vehicle control weekly for a period of 21 days (QW*3). The size of the tumor in each mouse is measured using calipers at the start of the treatment (Day 0) and again on Days 3, 7, 11, 14, 18 and 21 of the 21-day treatment period. The mean and standard error of the mean in tumor area is then calculated and plotted for each treatment group (Figure 8D).The results show that the average size of tumors in mice receiving both SI-77P26 and Sl- 77E14 was smaller than that in the vehicle control group of mice, with significant differences observed in drug vs vehicle treatment group at Day 7 through Day 21. By Day 21, average tumor volume increased from Day 0 by a factor of 11.5-fold in the vehicle treated group, 0.15-fold in the SI-77P26 group, and 0.06-fold in SI-77E14. Thus, in the absence of a 4-1BB binding domain, the tetraGNC antibody (P4-α4-lBB-null) can achieve the same level of tumor suppression as the pentaGNC antibody does.P2-αCD3, P4-α4-lBB-null tetraGNC antibodiesTo compare the positional effect of αC D3 at Pl and P2 position in the same in vivo assay, a pair of P2-αCD3 pentaGNC and tetraGNC antibodies, SI-77P37 and SI-77PE13, respectively, were generated and characterized. NOG mice received Fadu tumor cell grafts and human PBMC, and were divided into 3 groups to receive: SI-77P37, which a P2-αCD3 pentaGNC antibody having a P4-α4-1BB (clone 466F3) domain (1.5 mg / kg dose); SI-77E13 , which is a P2-αCD3 tetraGNC antibody without an lBαB4 a-t P4 position at the molecular equivalent of SI-77P37 (1.286 mg / kg), and a vehicle control (0.9% sodium chloride), respectively. Mice were dosed intravenously with drug or vehicle control weekly for a period of 21 days (QW*3). The size of the tumor in eachmouse is measured using calipers at the start of the treatment (Day 0) and again on Days 3, 7, 11, 14, 18 and 21 of the 21 -day treatment period. The mean and standard error of the mean in tumor area is then calculated and plotted for each treatment group (Figure 8E).The results show that the average size of tumors in mice receiving both SI-77P37 and SI- 77E13 was smaller than that in the vehicle control group of mice, with significant differences observed in drug vs vehicle treatment group at Day 7 through Day 18. By Day 18, average tumor volume increased from Day 0 by a factor of 11.5-fold in the vehicle treated group, 0.2-fold in the SI-77P37 group, and 0.01-fold in SI-77E13. Thus, in the absence of a 4-1BB binding domain, the P2-αCD3 tetraGNC antibody (P4-α4-lBB-null) can achieve the same level of tumor suppression as the P2-αCD3 pentaGNC antibody does.Pl-αCD3 versus P2-αCD3 and tetraGNC versus pentaGNC antibodiesThe Pl-αCD3 and P2- αCD3 GNC antibodies showed subtle differences in resting T cell internalization (see Example 6). The results in Figure 8D and 8E show that not only does P4-α4- 1BB not affect GNC function, but also the position of αCD3 at P 1 or P2 does not affect the function of tetraGNC and pentaGNC antibodies in tumor suppression. Pairwise comparisons showed no significant differences between the xenograft model systems under the same conditions. By the definition of T-cell engagers, SI-77X2 is a trispecific GNC T-cell engager, while SI-77E13 and SI-77E14 are tetraspecific GNC T-cell engagers, as SI-77X2 targets one TAA (EGFR) whereas SI-77E13 and SI-77E14 target two (i.e., EGFR and HER3). The three antibodies have similar tumor suppressor effects, suggesting that this in vivo xenograft system may be insufficient to distinguishing between αC D3 targeting and / or positional effects.Example 7. GNC T cell engagers with a PD-L1 binding domain at either P5 or P6 position, combined with a CD3 binding domain at P2, consistently produce the highest frequencies of polyfunctional CD4+and CD8+T cellsTo further evaluate the positional effect of GNC T cell engagers with various configurations of binding domains, the production of polyfunctional CD4+ T cells and CD8+ T cells were used to compare the efficacy by each T cell engagers of a distinct configuration. 20 GNC T cell engagers targeting 4 TAAs, namely HER2, GPC3, HLA-G, or uPAR were designed as shown in Table 4 and Figure 9. Each antibody was constructed, produced, and characterized according to the protocols as described in Example 2. Each group of antibodies comprise:(1) a CD3 -binding domain, positioned at either Pl or P2 Position of the heavy chain; and(2) a PD-L1 -binding domain, positioned at P3, P5, or P6 Position 3 of the antibody monomer. Next, the assay was performed in the absence of tumor cells or exogenous antigen. This configuration was chosen to isolate the effect of CD3 and PD-L1 domain positioning on T cell priming and polyfunctional cytokine production when T cells are stimulated only through baseline alloreactivity with monocytes.Human T cells were purified from PBMCs of a first donor, and monocytes were prepared from PBMCs of an unrelated donor. “PanT-Monocyte” co-cultures were established at a 2: 1 T cell monocyte ratio in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin. GNC molecules were added at multiple concentrations in a dose-response format and incubated for 24 hours at 37°C and 5% CO2. To enable intracellular cytokine detection, brefeldin A and monensin were added during the final 5 hours of culture to block cytokine secretion. At endpoint, cells were stained with a viability dye and surface lineage markers (TCRα / β, CD4, CD8), then fixed, permeabilized, and stained intracellularly for Granzyme-B, IFN- y, TNFa, and IL-2. Data analysis used a gating strategy of singlets — > live cells — > TCRα / β+T cells —> CD4+or CD8+subsets — cytokine-positive cells. Polyfunctional T cells were defined as cells co-expressing IFNy, TNFa and IL-2.GNC T cell engagers with a PD-Ll-binding domain at P5 or P6 position, combined with a CD3-binding domain in P2 position, consistently produced the highest frequencies of polyfunctional T cells across GNC targeting multiple TAA. Specifically, production of fFNy, Granzyme B, and polyfunctional T cell production of IFNy, TNFa and IL-2 were measured and compared as shown in Figure 10A, 10B, and 10C, respectively, and results were summarized in Table 5. The advantage of PD-L1 binding at P5 position was top ranked in both polyfunctional CD4+ and CD8+ T cell production when targeting HER2, whereas the advantage of PD-L1 binding at P6 position were top ranked in both polyfunctional CD4+ and CD8+ T cell production when targeting GPC3, HLA-G, or uPAR. When the CD3 binding was positioned in Pl position, differences in the positional effect between PD-L1 positions were reduced or absent.The results indicate that the presence of PD-L1 binding improves the efficacy of classic T cell engagers of anti-TAA and CD3 and that an optimized position of TAA, CD3 and PD-L1 in the antibody structure significantly improves the production and efficacy of polyfunctional T cells. The interplay among these binding domains strongly influences the ability of a T cell engager to prime cytokine-polyfunctional T cells in a monocyte co-culture lacking tumor antigens. Engagement of PD-L1 molecules by the PD-Ll-binding arm of the GNC anchored the molecule to the monocyte surface and blocked inhibitory PD-1 / PD-L1 signaling. When CD3 was in P2 position and PD-L1 was in either P5 or P6 position, the geometry of the complex appeared to favor optimal TCR engagement, producing maximal polyfunctional cytokine responses. This positional effect was generally consistent and independent of any TAA binding, highlighting a generalizable design principle for CD3 / PD-L1 multispecifics.Polyfunctional T cells can perform multiple functions simultaneously, particularly those that can produce multiple cytokines and degranulate lytic enzymes are associated with more effective control of infections and potentially cancer. In the context of cancer, polyfunctional T cells, especially CD4 T cells, can enhance tumor regression and promote long-term persistence when used in adoptive immunotherapy.The application discloses a therapeutic approach for enhancing T cell polyfunctionality, thereby improving patient outcomes in T cell-based cancer immunotherapies. By promoting robust anti-tumor activity, improving T cell proliferation and survival, coordinating immune responses, targeting heterogeneous tumors, reducing immune escape, and supporting memory formation, this therapeutic agent plays a vital role in achieving superior therapeutic outcomes for cancer patients.A key aspect of GNC T cell engager discovery is their ability to improve the quality of T cells in the patient in addition to directing their interaction with cancer cells. This enhancement of T cell quality significantly boosts the immune response against tumors. The improvement in T cell quality is achieved through the structural features of GNC T cell engagers that provide polyfunctional cytokine activity upon interaction with tumor cell or other cells, such as monocytes. The polyfunctional cytokine activity of GNC T cell engagers involves the interaction with monocytes through the PD-L1 domain at position 5 or 6. This interaction induces the release of a broad range of cytokines by T cells, which play various roles in promoting T cell activation, proliferation, and survival. The cytokines released enhance the functional quality of T cells, improving their cytotoxic capabilities and resistance to exhaustion. This pre-conditioning of T cells prepares them for more effective engagement with cancer cells.The structural features of GNC T cell engagers include a modular design that allows for the independent functioning of different domains, such as regions for monocyte interaction and cytokine induction, separate from the TAA binding domains. The cytokine induction domain within the GNC structure is responsible for inducing polyfunctional cytokine activity upon monocyte interaction, triggering a cascade of immune-modulating signals that enhance overall immune function. This design provides flexibility in TAA binding, ensuring that the absence of TAA binding does not hinder the ability of GNC T cell engagers to improve T cell quality via monocyte interaction. This flexibility allows for broader application and effectiveness in diverse immune environments.The ability of GNC T cell engagers to improve T cell quality before targeting cancer cells is particularly valuable in clinical settings. This pre-conditioning of T cells can lead to a more robust and sustained anti-tumor response, especially in patients with compromised or exhausted T cell populations. By leveraging the structural features that promote polyfunctional cytokine activity, GNC T cell engagers create a more favorable immune landscape, enhancing the overall effectiveness of cancer immunotherapy.The application therefore provides a novel approach to cancer treatment, utilizing the multi-modal activities of GNC T cell engagers to improve patient outcomes through enhanced T cell quality and targeted tumor cell elimination.TABLESTable 1. Configurations of GNC T-Cell Engagers characterized by having aPD-Ll at P5.Table 2A. The expression and stability of pentaGNC antibodies. % protein of interest (%POI) from UPLC-SEC traces were calculated after proA elution and 1 month post purification.Table 2B. The domain affinity and avidity of pentaGNC antibodies binding to each human antigen.Table 3. The stability and binding affinity of anti-DLL3 GNC T-cell engagers. % protein of interest (%POI) from UPLC-SEC traces were calculated after proA elution.Table 4. Configurations of GNC T-Cell Engagers characterized by having ocPD-Ll at P5 or P6.Table 5. GNC T cell engagers with a PD-L1 binding domain at either P5 or P6 position, combined with a CD3 binding domain at P2, consistently produce the highest frequencies of polyfunctional CD4+and CD8+T cells when a TAA binding domain at Pl position engaging the binding to a tumor cell (ranked from 1 to 6, 1 being the highest). MU = Molecule is Unavailable.SEQUENCESThe sequence listed herein include nucleic acid and amino acid sequences related to representative embodiments of the application. For ease of reference, complementarity-determining region (CDR) sequences are indicated by underlining within the sequences. The underlining is provided solely as a visual aid to identify these regions in the present disclosure and does not define or limit the scope of the application. Unless otherwise specified, residue numbering follows the conventional numbering schemes applicable to the sequence type.

Claims

GNC T-CELL ENGAGER AND METHOD OF MAKING AND USING THEREOFCLAIMSWhat is claimed is:

1. A multi-specific antibody monomer having a light chain (LC) and a heavy chain (HC), each having a N-terminal and a C-terminal, comprising, a first scFv domain having a binding affinity to a tumor-associated antigen (TAA) at the N- terminal of the HC (Position 1, or Pl), a Fab domain having a binding affinity to CD3, and a second scFv domain having a binding affinity to PD-L1 at the C-terminal of the LC (Position 5, or P5) or the N-terminal of the LC (Position 6, or P6).

2. The multi-specific antibody monomer of Claim 1, wherein the TAA comprises EGFR, HER3, DLL3, HER2, GPC3, HLA-G, uPAR, Nectin4, Fra, Tissue Factor, B7-H4, VEGFR2, B7- H3, Mucl6, CEACAM6, Claudin6, CLDN18.2, LGR5, GCC, CD20, CD19, Integrin 6, Mucl, CDH6 , FAP, CAIX, Integrin 04, CDH17, Lewis B / Y, GPRC5D, CEACAM5, ROR1, EGFR vIII.

3. The multi-specific antibody monomer of Claim 1, wherein the Fab domain comprises complementarity-determining regions (CDRs) having the amino acid sequences comprising:VL CDR1: ESISSWLA;VL CDR2: EASKLASGVPS;VL CDR3: QGYFYFISRTYVNS; VH CDR1 : GFTISTNAMS;VH CDR2: VITGRD1TYYASWAKG; andVH CDR3: DGGSSAITSNNI.

4. The multi-specific antibody monomer of Claim 1, wherein the second scFv domain comprises the CDRs having the amino acid sequences comprising:VL CDR1: QSISSHLN;VL CDR2: KASTLASGVPS;VL CDR3: QQGYSWGNVDNV;VH CDR1 : GFSFSSGYDMC;VH CDR2: CIAAGSAGITYDANWAKG; and VH CDR3 : S AF SFD YAMDL .

5. The multi-specific antibody monomer of Claim 1, further comprising a third scFv domain at the C-terminal of the HC (Position 3, P3).

6. The multi-specific antibody monomer of Claim 3, further comprising a fourth scFv domain attached to the third scFv domain (Position 4, P4) through a linker.

7. The multi-specific antibody monomer of Claim 1, further comprising a fifth scFv domain at the P5 or P6.

8. The multi-specific antibody monomer of Claim 5, wherein the third scFv domain has a binding affinity to PD-L1, HER3, or 4-1BB.

9. The multi-specific antibody monomer of Claim 6, wherein the fourth scFv domain has a binding affinity to 4-1BB or FITC.

10. The multi-specific antibody monomer of Claim 1, wherein the antibody monomer is trispecific, tetra-specific, penta-specific, or hexa-specific.

11. The multi-specific antibody monomer of Claim 1, wherein the antibody monomer is trispecific and wherein the TAA comprises DLL3, EGFR, or HER2.

12. The multi -specific antibody monomer of Claim 5, wherein the antibody monomer is tetra- specific, and wherein the third scFv domain has a binding affinity to HER3, 4- IBB, or PD-L1.

13. The multi-specific antibody monomer of Claim 12, wherein the second scFV domain is at P5.

14. The multi-specific antibody monomer of Claim 12, wherein the second scFV domain is at P6.

15. The multi-specific antibody monomer of Claim 6, wherein the antibody monomer is penta- specific, and wherein the third scFv domain has a binding affinity to HER3 or PD-L1, and the fourth scFv domain has a binding affinity to 4-1BB.

16. The multi-specific antibody monomer of Claim 15, wherein the second scFv domain is at P5.

17. The multi-specific antibody monomer of Claim 15, wherein the second scFv domain is atP6.

18. The multi-specific antibody monomer of Claim 1, having a binding affinity to CD3, PD- Ll, and DLL3.

19. The multi-specific antibody monomer of Claim 1, having a binding affinity to CD3, PD- Ll, a TAA selected from EGFR, DLL3, HER2, GPC3, HLA-G, and uPAR, and one of HER3 and 4-1BB.

20. The multi-specific antibody monomer of Claim 1, having a binding affinity to CD3, PD- Ll, EGFR, HER3, and 4- IBB.

21. A monoclonal antibody, comprising the multi-specific antibody monomer of any one of Claim 1-20.

22. The monoclonal antibody of Claim 21, wherein the antibody is tri-specific, tetra-specific, penta-specific, or hexa-specific.

23. An isolated nucleic acid sequence encoding the multi-specific antibody monomer of any one of Claim 1-18, or the monoclonal antibody of Claim 21 or 22.

24. An expression vector comprising the isolated nucleic acid sequence of Claim 23.

25. A host cell comprising the isolated nucleic acid sequence of Claim 24.26 A method for producing the monoclonal antibody of Claim 21, comprising culturing a host cell such that the DNA sequence encoding the monoclonal antibody is expressed, and purifying said monoclonal antibody.

27. A method of making the monoclonal antibody of Claim 21, comprising culturing a host cell under conditions wherein said monoclonal antibody is produced, and recovering said monoclonal antibody.

28. An immunoconjugate comprising the monoclonal antibody of Claim 21 covalently linked to a cytotoxic agent.

29. A pharmaceutical composition, comprising the monoclonal antibody Claim 21 or the immunoconjugate of Claim 28, and optionally a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of Claim 29, further comprises a therapeutic agent, wherein the therapeutic agent comprises an antibody, an immunoconjugate, a chemotherapeutic agent, a toxin, a radionuclide, or a combination thereof.

31. A method for treating or preventing a cancer, an autoimmune disease, or an infectious disease in a subject, said method comprising administering to the subject an effective amount of the monoclonal antibody of Claim 21 or the immunoconjugate of Claim 28.

32. The method of Claim 31, wherein the monoclonal antibody is administered intrathecally, intraperitoneally, or by convection-enhanced delivery.

33. The method of Claim 32, further comprises co-administering a therapeutic agent, wherein the therapeutic agent comprises an antibody, an immunoconjugate, a chemotherapeutic agent, a toxin, a radionuclide, or a combination thereof.

34. A solution comprising an effective concentration of the monoclonal antibody of Claim 21, wherein the solution is blood plasma in a subject.

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