Guidance and navigation control proteins, methods for producing the same, and methods for using the same

Abstract

To provide methods of making and using guidance and navigation control (GNC) antibodies with multiple binding activities against surface molecules of immune cells and tumor cells.SOLUTION: The application provides a multi-specific antibody-like protein having a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, a first binding domain (D1) at the N-terminal, a second binding domain (D2) comprising a light chain moiety, a Fc region, a third binding domain (D3), and a fourth binding domain (D4) at the C-terminal, the light chain moiety comprising a fifth binding domain (D5) covalently attached to the C-terminal and / or a sixth binding domain (D6) covalently attached to the N-terminal, and the D1, D2, D3, D4, D5 and D6 each having a binding specificity against a tumor antigen, an immune signaling antigen, or a combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 931,307 filed on 6 November 2019, U.S. Provisional Application No. 62 / 984,731 filed on 3 March 2020, and U.S. Provisional Application No. 62 / 991,042 filed on 17 March 2020 (35 U.SC 119(e)), the entire disclosure of which is incorporated herein by reference.

[0002] This application relates to the technical field of multispecific antibodies used in immunotherapy, and more specifically, to methods for producing and using guidance and navigation control (GNC) antibodies that have multiple binding activity to surface molecules of immune cells and tumor cells. [Background technology]

[0003] Cancer progresses by acquiring mutations that enable cancer cells to transform, proliferate, and metastasize while evading immune surveillance and response. Antibody therapies for treating cancer employ several different mechanisms. For example, monoclonal antibodies targeting growth factor receptors (EGFR, HER2, etc.) overexpressed on tumor cells can be used to block tumor cell proliferation. Blocking inhibitory T cell checkpoint signals (anti-PDL1, anti-PD1, anti-CTLA4) using antibodies is a method of preventing tumor cells by weakening the immune response that controls tumor cell proliferation. Another method is to inhibit angiogenesis (e.g., anti-VEGF), which slows tumor cell growth by reducing access to oxygen and nutrients. Monoclonal antibodies and antibody-drug conjugates (ADCs) are initially effective in controlling tumors. However, cancer resistance to antibody therapy often arises from evasion mechanisms such as loss of the external domain, receptor downregulation, and receptor mutation (Miller et al. Clin Cancer Res. 2017; Reslan et al. Mabs. 2009; Loganzo et al. Mol Cancer Ther. 2016). For example, resistance to the anti-HER2 mAb trastuzumab may arise from ectodomain shedding of HER2 or occlusion of the trastuzumab epitope on HER2 (Fiszman and Jasnis. International Journal of Breast Cancer, 2011).

[0004] Combination therapy, which combines multiple therapeutic mechanisms such as chemotherapy, radiotherapy, and antibody therapy, has become the mainstream treatment strategy. In this specification, multispecific antibodies are used to combine different antibody therapies and mechanisms into a single drug (Boumahdi and de Sauvage. Nat Rev Drug Discov. 2020). [Overview of the project]

[0005] The following summary is illustrative and not intended to be limiting. Further embodiments, features, and characteristics beyond those described above will become apparent by reference to the drawings and the detailed description below.

[0006] In one embodiment, the present invention provides a guidance and navigation control (GNC) protein that can simultaneously bind to effector cells and target cells. The GNC protein may be a monomer or a dimer of a monomer. The GNC protein may be an antibody or an antibody-like protein. The GNC protein may have at least five or six binding domains.

[0007] In one embodiment, the present invention provides a multispecific antibody-like protein having an N-terminus and a C-terminus, comprising, sequentially from the N-terminus to the C-terminus, a first binding domain (D1) located at the N-terminus, a second binding domain (D2) including a light chain portion, an Fc region, a third binding domain (D3), and a fourth binding domain (D4) located at the C-terminus. The light chain portion includes a fifth binding domain (D5) covalently bonded to the C-terminus and / or a sixth binding domain (D6) covalently bonded to the N-terminus. D1, D2, D3, D4, D5, and D6 each have binding specificity to a tumor antigen, an immune signaling antigen, or a combination thereof.

[0008] Tumor antigens may be tissue antigens, neonatal antigens, tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), or combinations thereof.

[0009] D2 is C H1 It may also include. In one embodiment, the light chain portion in D2 is C L It may also include. In one embodiment, the light chain portion is C κ / C λ It may include.

[0010] D2 may contain a dimer.

[0011] In one embodiment, D2 may comprise a Fab region. In one embodiment, the Fab region comprises V L and V H may have a disulfide bond therebetween. In one embodiment, D2 comprises V L and V H may be included.

[0012] In one embodiment, D2 may comprise a receptor. In one embodiment, the receptor may be NKG2D. In one embodiment, D2 is C H1 and C L may comprise NKG2D bound to. In one embodiment, D2 may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NOs: 155 and 116.

[0013] D2 may be bound to an Fc region via a hinge.

[0014] The Fc region may comprise null mutations that can reduce or eliminate effector function. In one embodiment, the Fc region may be a wild-type Fc. In one embodiment, the region may comprise LALAKA mutations for null Fc. In one embodiment, the LALAKA mutation for null Fc may comprise the L234A / L235A / K322A (Eu numbering) mutation. In one embodiment, the Fc region may comprise a G237A (Eu numbering) mutation. In one embodiment, the Fc region may comprise an N297A (Eu numbering) mutation. In one embodiment, the Fc region may comprise a glycosylated Fc. In one embodiment, the Fc region may be a glycosylated Fc that reduces effector function.

[0015] In one embodiment, according to the present invention, there is provided a multispecific antibody-like protein having an N-terminus and a C-terminus, sequentially from the N-terminus to the C-terminus: a first binding domain (D1) located at the N-terminus, and C L and C H1 a second binding domain (D2) comprising a dimer bound to, and C via a hinge H1A multispecific antibody-like protein is provided, comprising an Fc region containing CH2 and CH3 bound to a third binding domain (D3) and a fourth binding domain (D4) located at the C-terminus. The light chain portion may have a fifth binding domain (D5) covalently bound to the C-terminus and / or a sixth binding domain (D6) covalently bound to the N-terminus. D1, D2, D3, D4, D5, and D6 may each have binding specificity to a tumor antigen, an immune signaling antigen, or a combination thereof.

[0016] The dimers in D2 are C L and C H V joined L and V H It may also include a pair. The D2 domain may be a Fab region. The GNC protein may be a multispecific antibody monomer or a multispecific antibody.

[0017] In one embodiment, the multispecific antibody-like protein may be quintuple or hexaple specific.

[0018] In one embodiment, the light chain portion of D2 may have a fifth binding domain (D5) covalently bonded to the C-terminus, and the multispecific antibody-like protein is quintuple specific. In another embodiment, the light chain portion may have a sixth binding domain (D6) covalently bonded to the N-terminus, and the multispecific antibody-like protein is quintuple specific. In yet another embodiment, the light chain portion may simultaneously have a fifth binding domain (D5) covalently bonded to the C-terminus and a sixth binding domain (D6) covalently bonded to the N-terminus. This results in a hexaple specific multispecific antibody-like protein.

[0019] D1, D2, D3, D4, D5, and D6 may each independently be an scFv domain, a receptor, or a ligand.

[0020] The scFv domain has a V shape from the N-terminus to the C-terminus. L V H or V H V LIt may have the following configuration. In one embodiment, the scFv domain may have the R19S(Kabat) mutation. In one embodiment, the scFv domain is V L and V H A disulfide bond may be included between them. In one embodiment, the disulfide bond is V of the scFv domain. L 100 and V H It may be located between 44 (Kabat). In one embodiment, the scFv domain may contain an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with sequence numbers 72-112.

[0021] In one embodiment, D1, D2, D3, D4, D5, and D6 may all be scFv domains.

[0022] In one embodiment, D1, D2, D3, D4, D5, and D6 may each be independently a receptor or ligand. In one embodiment, at least one, two, three, four, or five of D1, D2, D3, D4, D5, and D6 may be receptors or ligands. In one embodiment, D1, D2, D3, D4, D5, and D6 may all be receptors or ligands. In one embodiment, D4, D5, or D6 may be a receptor or ligand. In one embodiment, the receptor or ligand may contain an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 113-116.

[0023] In one embodiment, D2 has binding specificity to CD3 or tumor-associated antigen (TAA).

[0024] In one embodiment, D1, D2, D3, D4, D5, and D6 each independently have binding specificity to antigens selected from T cell receptors, immune checkpoint receptors, costimulatory receptors, lymphocyte or myeloid cell receptors, tumor-associated antigens (TAAs), tissue antigens, nascent antigens, tumor-specific antigens (TSAs), glycoproteins, and combinations thereof.

[0025] In one embodiment, the binding domain for the receptor on the T cell may be adjacent to the binding domain for the tumor-associated antigen (TAA). In one embodiment, the binding domain for the receptor on the T cell may be adjacent to the binding domain for the receptor on the lymphocyte or myeloid cell.

[0026] In one embodiment, the receptor on the T cell may be CD3, a T cell receptor, or a complex thereof. In one embodiment, the immune checkpoint receptor may be PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, CD160, LOX-1, siglec-15, CD47, SIRPα, or a combination thereof. In one embodiment, the costimulatory receptor may be 4-1BB, CD28, OX40, GITR, CD40, ICOS, CD27, CD30, CD226, or a combination thereof. In one embodiment, the tumor-associated antigen (TAA) may be EGFR, HER2, HER3, HER4, EGRFVIII, CD19, claudin 18.2, BCMA, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, cMET, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TACI, TROP2, NKG2D ligand, PD-L1, or a combination thereof.

[0027] In one embodiment, D1, D2, D3, D4, D5, and D6 may each independently have binding specificity to antigens selected from EGFR, HER2, HER3, EGFRvIII, ROR1, CD3, CD28, CEA, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D ligand, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73, and CD39, CLDN18.2, and CSF1R. The Fc region includes the human IgG Fc region.

[0028] In one embodiment, D2 and D5 each independently have binding specificity to a tumor-associated antigen, a neonatal antigen, or a tumor-specific antigen (TSA).

[0029] In one embodiment, D1 has binding specificity to CD3, CD20, EGFR, or derivatives thereof. In one embodiment, D2 has binding specificity to EGFR, CD3, HER2, MSLN, NKG2D ligand, or derivatives thereof. In one embodiment, D3 has binding specificity to PD-L1. In one embodiment, D4 may contain a 4-1BBL trimer or have binding specificity to 4-1BB or derivatives thereof. In one embodiment, D5 has binding specificity to HER3, CD19, NKG2D ligand, or derivatives thereof. In one embodiment, D6 has binding specificity to CD19.

[0030] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 has binding specificity to EGFR, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D5 has binding specificity to HER3. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 1-8.

[0031] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD20, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 9-12.

[0032] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD20, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D5 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 13-16.

[0033] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 has binding specificity to MSLN, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D5 has binding specificity to NKG2D ligand. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 17-20.

[0034] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 has binding specificity to HER2, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D5 has binding specificity to NKG2D ligand. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 21-24.

[0035] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 25-28.

[0036] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 contains a 4-1BB ligand trimer, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 29-32.

[0037] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 33-36.

[0038] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 contains a 4-1BB ligand trimer, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 37-40.

[0039] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 has binding specificity to EGFR, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 41-44.

[0040] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 45-48.

[0041] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 contains a 4-1BB ligand trimer, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 49-52.

[0042] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 contains NKG2D, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to EGFR. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 117-120.

[0043] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 contains NKG2D, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 123-126.

[0044] In one embodiment, the multispecific antibody-like protein is quintuplicate, where D1 has binding specificity to CD3, D2 contains NKG2D, D3 has binding specificity to PD-L1, D4 contains a 4-1BB ligand trimer, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 127-130.

[0045] In one embodiment, the multispecific antibody-like protein is hexaspecific, where D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, D5 has binding specificity to HER3, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 53-60.

[0046] In one embodiment, the multispecific antibody-like protein is hexaspecific, where D1 has binding specificity to CD3, D2 has binding specificity to EGFR, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, D5 has binding specificity to HER3, and D6 has binding specificity to CD19. In one embodiment, the multispecific antibody-like protein contains an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 61-68.

[0047] In one embodiment, D1, D3, D4, D5, or D6 is (G x S y ) n A linker may be included. n may be an integer from 1 to 10. x may be an integer from 1 to 10. y may be an integer from 1 to 10.

[0048] In one embodiment, the present invention provides a guidance navigation control (GNC) protein comprising the multispecific antibody-like protein described herein. In one embodiment, such a GNC protein may be a dimer of the multispecific antibody-like protein described herein.

[0049] In one embodiment, the present invention provides an isolated nucleic acid sequence encoding an amino acid sequence of a multispecific antibody-like protein, a fragment thereof, or a derivative thereof as described herein.

[0050] In one embodiment, the present invention provides an expression vector containing the above-mentioned isolated nucleic acid sequence.

[0051] In one embodiment, the present invention provides a host cell containing the above-mentioned isolated nucleic acid sequence. In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell.

[0052] In one embodiment, the present invention provides a method for producing the above-mentioned GNC protein. In one embodiment, the method for producing a multispecific antibody or monomer includes the steps of culturing a host cell containing an isolated nucleic acid sequence such that a DNA sequence encoding the multispecific antibody or monomer is expressed, and purifying the multispecific antibody. The isolated nucleic acid sequence encodes the amino acids of the multispecific antibody-like protein.

[0053] In one embodiment, the present invention provides an immunocomplex comprising a cytotoxic agent or imaging agent conjugated via a linker to a GNC protein such as a multispecific antibody-like protein or a multispecific antibody. The linker may include covalent bonds, such as ester bonds, ether bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, hydrophobic poly(ethylene glycol) linkers, or combinations thereof.

[0054] In one embodiment, the cytotoxic agent or imaging agent may be a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent derived from the calicheamicin class, an antimitotic agent, a toxin, a radioisotope, a therapeutic agent, or a combination thereof.

[0055] In one embodiment, the present invention provides a pharmaceutical composition for treating, preventing, or controlling a medical condition such as cancer, autoimmune disease, or infectious disease. In one embodiment, the composition may comprise a pharmaceutically acceptable carrier, a GNC protein, for example, a multispecific antibody or multispecific antibody-like protein, an immune complex thereof, or fragments thereof.

[0056] In one embodiment, the pharmaceutical composition may further include a therapeutic agent selected from radioisotopes, radionuclides, toxins, chemotherapeutic agents, or combinations thereof.

[0057] In one embodiment, the present invention provides a method for treating, preventing or controlling a medical condition such as cancer, autoimmune disease, or infectious disease. In one embodiment, the method includes the step of administering a pharmaceutical composition comprising a purified multispecific antibody, a multispecific antibody-like protein, or a fragment thereof.

[0058] In one embodiment, the present invention provides a method for treating a human subject having cancer, an autoimmune disease, or an infectious disease. In one embodiment, the method includes administering an effective amount of GNC protein, for example, purified multispecific antibodies, multispecific antibody-like proteins, or fragments thereof, to the subject.

[0059] In one embodiment, the above method may further include the step of co-administering an effective amount of therapeutic agent. The therapeutic agent includes antibodies, chemotherapeutic agents, enzymes, anti-estrogen agents, receptor tyrosine kinase inhibitors, kinase inhibitors, cell cycle inhibitors, checkpoint inhibitors, DNA, RNA or protein synthesis inhibitors, RAS inhibitors, inhibitors of PD1, PD-L1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, or combinations thereof.

[0060] In one embodiment, the present invention provides a solution containing an effective concentration of GNC protein, such as a multispecific antibody, a multispecific antibody-like protein, or a fragment thereof. In one embodiment, the above solution may be the plasma of a human subject. [Brief explanation of the drawing]

[0061] The aforementioned and other features of the disclosure herein will become more fully apparent from the following description and the appended claims, together with the accompanying drawings. The drawings show only a few embodiments prepared in accordance with the disclosure herein and should therefore not be considered limiting, and further specificities and details of the disclosure herein may be illustrated through the use of the accompanying drawings.

[0062] [Figure 1] The schematic structure of the antigen-binding domain is shown. Figure 1A is a penta-GNC antibody, and Figure 1B is a hexa-GNC antibody. In Figure 1, the variable region of Fab (substitutable with receptor or ligand) is black (D2), the constant region and Fc region of Fab are white, and the additional scFv antigen-binding domain is a shaded box (each substitutable with receptor-ligand binding). The heavy chain monomer has D1 bound to its N-terminus, and D3 and D4 bound to its C-terminus via D4. The light chain monomer has D5 and / or D6 bound to its N-terminus and C-terminus. [Figure 2] In the presence of human pancreatic cancer cells (BxPC3) expressing high levels of EGFR and low levels of HER3, the penta-GNC antibody (SI-1P1) exhibits maximum T cell activation and demonstrates similar efficacy to tetra-GNC antibodies targeting HER3 (tetra) or non-tumor antigens (tetra, FITC) and bispecific antibodies targeting only tumor antigens (BI). [Figure 3]The high efficacy of SI-1P1 is demonstrated in TDCC assays using cancer cell lines expressing high levels of EGFR and low levels of HER3 in human breast cancer cells (MDA-MB-231) (Figure 3A) and human cervical cancer cells (HeLa) (Figure 3B), as well as control antibodies (including a similar tetra-GNC antibody lacking binding to HER3, a tetra-GNC control antibody lacking binding to both tumor antigens, and a bispecific antibody targeting only the tumor antigen). [Figure 4] This demonstrates the effectiveness of having the NKG2D receptor as a binding domain for GNC antibodies. Figure 4A shows the efficacy of SI-49P3-mediated T cell activation using human pancreatic cancer cells (BxPC3). Figure 4B shows the high efficacy of SI-49P1 in a TDCC assay using human breast cancer cells (MDA-MB-231) expressing tumor antigens other than EGFR and HER3 (MICA and mesothelin), and two control antibodies (a tetra-GNC antibody lacking NKG2D, and a tri-GNC antibody lacking binding specificity to both PD-L1 and 4-1BB). [Figure 5] In a reporter bioassay using Jurkat cells, the 4-1BBL-trimer-Fc fusion protein mediates stronger activation of 4-1BB signaling than other molecules including monomer 4-1BB ligand, monomer Fc, or anti-4-1BB scFv. [Figure 6] Octet binding analysis of pentaGNC antibodies compared to humanized anti-huEGFR domains is shown. Variants of the humanized EGFR-binding domain (H1, H4, or H7), whether as the scFv domain in SI-55P3, SI-79P2, SI55P9, and SI-79P3, or as Fab in SI-77P1, maintain tight binding to human EGFR with minimal regio-effects. [Figure 7] The efficacy and EC50 values ​​of the pentaGNC antibody in a TDCC assay using human pancreatic cancer cells (BxPC3) as target cells are shown (Figure 7A: SI-1P1, 0.2814 pM; Figure 7B: SI-55P9, 0.4871 pM; Figure 7C: SI-55P10, 0.7358 pM). [Figure 8]This study demonstrates the efficacy of pentaGNC proteins containing NKG2D at position D2 in a TDCC assay using human breast cancer cells (MDA-MB-231; expressing MICA) as target cells. The resulting EC50 values ​​were SI-49P6, 0.7366 pM, and SI-49P7, 0.1094 pM. [Figure 9] The tetra-GNC antibody SI-35E20 induces RTCCs into transduced lung cancer cells A549 using NucRed, and suppresses the proliferation of lung adenocarcinoma cells in the presence of PBMCs. [Figure 10] The tetra-GNC antibody SI-38E17 demonstrates that it induces RTCCs to Nuc-GFP Nalm-6 leukemia cells in the presence or absence of donor PBMCs. [Figure 11] The tetra-GNC antibody SI-39E18 demonstrates that it induces RTCC in the presence of donor PBMCs compared to a solvent control, killing NucRed+ UMUC3viii cells derived from human bladder cancer. [Figure 12] By administering JVM-3 cells and donor PBMCs (5 × 10⁶ and 2 × 10⁷, respectively) to NCG mice, the tetra-GNC antibody SI-38E17 effectively suppresses the growth of human B-cell leukemia cells in a human tumor xenograft model. [Figure 13] By administering UM-UC-3-EGFR VIII cells and human PBMCs (5 × 10⁶ and 5 × 10⁶, respectively) to NCG mice, the tetra-GNC antibody SI-39E18 effectively suppresses the growth of human bladder cancer cells in a human tumor xenograft model. [Figure 14] In the TDCC assay, the hexa-GNC antibody (SI-55H11) mediates more complete elimination of human cervical cancer cells (Hela) compared to similar trispecific antibodies, suggesting the need to target immunomodulatory proteins (e.g., PD-L1 immune checkpoint) while simultaneously activating 4-1BB. [Figure 15]In a TDCC assay using human pancreatic cancer cells (BxPC3) expressing low levels of HER3, the hexa-GNC antibody (SI-55H11) demonstrated improved efficacy compared to the parent penta-GNC antibody (SI-55H9) by binding more effectively to HER3. [Modes for carrying out the invention]

[0063] The following detailed description refers to the accompanying drawings, which constitute part of it. In the drawings, unless otherwise indicated in the context, similar symbols generally identify similar components. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. As described herein and shown in the figures, it will be readily apparent that aspects of this disclosure can be arranged, substituted, combined, separated, and designed in the various configurations expressly considered herein.

[0064] The present invention relates to guidance and navigation control (GNC) proteins, methods for producing the same, and methods for using the same. In some embodiments, the GNC protein may be a multispecific antibody-like protein. In some embodiments, the GNC protein may be a multispecific antibody. In this case, the GNC protein is also called a GNC antibody. In some embodiments, the present invention provides a quintuple-specific antibody-like protein and a hexaple-specific antibody-like protein. In some embodiments, the present invention provides a quintuple-specific antibody and a hexaple-specific antibody.

[0065] GNC proteins include proteins that bind multiple functionally independent binding sites to a single entity that yields both effector and target cells (see applicant's application WO / 2019 / 005642, which is incorporated herein by reference in its entirety). In one embodiment, these multispecific binding molecules targeting tumor antigens and immune-activating receptors can utilize mechanisms similar to immune effector cell-mediated tumor death at minimal cost. Rather than genetically modifying individual patient T cells, such multispecific binding molecules can be efficiently manufactured on a large scale and administered by more common, off-the-shelf methods. Among GNC proteins, polyspecific antibodies, such as quadruspecific antibodies, have been shown to exert desirable multifaceted GNC effects, possessing structurally and functionally diverse but relatively independent binding domains (see applicant's application WO / 2019 / 191120, which is incorporated herein by reference in its entirety).

[0066] In one embodiment, the GNC protein may comprise a multispecific antibody-like protein comprising a heavy chain portion and a light chain portion. The Fab region of the antibody consists of one constant domain and one variable domain derived from the heavy chain portion and the light chain portion. The heavy chain may comprise three additional antigen-specific binding domains bound to the N-terminus and / or C-terminus. The light chain portion may comprise one or two additional binding domains bound to the N-terminus and / or C-terminus.

[0067] In some embodiments, the GNC antibody may be a penta-GNC antibody or a hexa-GNC antibody, as shown in Figure 1. The GNC antibody may have the ability to induce immune cells (or other effector cells) to tumor cells (or other target cells) via the binding of multiple surface molecules on immune cells and tumor cells. The immune cells may be cells of the human immune system. Cells of the human immune system include, but are not limited to, leukocytes, peripheral blood mononuclear cells (PBMCs), T cells, and natural killer cells (NK cells). Other target cells include, but are not limited to, autoimmune cells (normal B cells), tissue target cells, non-tumor cells, infected cells, inflammatory cells, and damaged cells. In some embodiments, the T cells include human T cells. Human T cells include, but are not limited to, naive T cells, activated T cells, helper T cells, regulatory T cells, memory T cells, and exhausted T cells. In one embodiment, the tumor cells express a tumor antigen. Tumor antigens include, but are not limited to, tumor-specific antigens (TSAs), neonatal antigens, and tumor-associated antigens (TAAs).

[0068] In one embodiment, the GNC antibody may include at least one binding domain capable of binding to one surface molecule on a T cell and at least one binding domain capable of binding to one surface antigen on a tumor cell (Table 1). In some embodiments, the surface molecule on the T cell includes, but is not limited to, a signaling protein, which includes, but is not limited to, CD3, NKG2D, and 4-1BB. The surface molecule on the NK cell includes, but is not limited to, a signaling protein, which includes, but is not limited to, NKG2D and 4-1BB. The surface antigen on the tumor cell includes, but is not limited to, a tumor antigen, which includes, but is not limited to, EGFR, HER2, HER3, MSLN, CD19, and PD-L1. In one embodiment, the tumor cell constitutes a tumor or cancer. This tumor or cancer includes, but is not limited to, solid tumors, sarcomas, hematopoietic malignancies, lung cancer, pancreatic cancer, bladder cancer, cervical cancer, breast cancer, leukemia, and lymphoma.

[0069] GNC antibodies with at least four additional binding domains in addition to D2 may require structural stability to maintain the independent function of binding specificity and affinity of each binding domain. Each additional binding domain is (G x S y ) n A peptide linker may be included. Here, n is an integer from 1 to 10, x is an integer from 1 to 10, and y is an integer from 1 to 10.

[0070] In one embodiment, the binding domain, for example, D1, D2, D3, D4, D5, or D6, may be a single-chain variable fragment (scFv), a receptor, or a ligand (Table 1). The scFv domain is V H -V L (HL) or V L -V H In either of the (LH) orientations, the heavy chain (V H ) and light chain (V L ) may be configured to have a fusion of variable regions. In one embodiment, the scFv domain is V H 44 and V L A stapled structure may be formed by introducing a disulfide bond between 100 (Kabat) units. In one embodiment, V on any of the light chain portions H3 V for containing scFv H The region contains the R19S mutation (Kabat numbering).

[0071] The binding domain may be configured to bind to at least one epitope of the antigen. The epitopes include, but are not limited to, CD3, 4-1BB, EGFR, HER2, HER3, MSLN, CD19, and PD-L1. The amino acid sequence selected to encode the anti-EGFR binding domain may be a humanized sequence. In other embodiments, the amino acid sequence selected to encode the anti-CD19 binding domain is a humanized sequence.

[0072] In one embodiment, the binding domain may be a receptor. In one embodiment, the receptor may be NKG2D. In one embodiment, D2 may include NKG2D.

[0073] The binding domain may be a ligand for the receptor, for example, 4-1BBL (4-1BB receptor ligand) and a 4-1BBL trimer for 4-1BB, or the receptor itself.

[0074] As used herein, the terms “a,” “an,” and “the” are defined to mean “one or more,” and include the plural form unless the context is appropriate.

[0075] The term "antibody" is used in its broadest sense and specifically covers single monoclonal antibodies (agonist antibodies and antagonist antibodies), antibody compositions having polyepitope specificity, and antibody fragments (e.g., Fab, F(ab′)2, and Fv) insofar as they exhibit desired biological activity. In some embodiments, the antibody may be monoclonal, chimeric, single-stranded, multispecific, multi-effective, human, and humanized antibodies. Examples of active antibody fragments that bind to known antigens include Fab, F(ab′)2, scFv, and Fv fragments, as well as products of Fab immunoglobulin expression libraries, and epitope-binding fragments of any of the antibodies and fragments described above. In some embodiments, the antibody may include immunoglobulin molecules and molecules containing immunoactive portions of immunoglobulin molecules, i.e., binding sites that bind immunospecifically to antigens. Immunoglobulins may be immunoglobulin molecules of any type (IgG, IgM, IgD, IgE, IgA, and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one embodiment, antibodies may be complete antibodies and any antigen-binding fragments derived from complete antibodies. A typical antibody usually refers to a heterotetramer protein consisting of two heavy (H) chains and two light (L) chains. Each heavy chain has a heavy chain variable domain (V H It consists of a light chain (abbreviated as) and a heavy chain constant domain. Each light chain portion is a light chain partial variable domain (V LIt consists of (abbreviated as) and a light chain sub-constant domain. H Region and V L The region can be further divided into domains of more conserved regions called hypervariable complementarity determination regions (CDRs) and framework regions (FRs). Each variable domain (V H or V L A carboxyl molecule typically consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding regions that interact with the antigen.

[0076] As used herein, the term “monoclonal” antibody includes “monoclonal monospecific,” “chimeric,” and “multispecific” antibodies (immunoglobulins), as well as fragments of these antibodies (insofar as they exhibit the desired biological activity), wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass (USPat. No. 4, 816, 567; Morrison et al., PNAS USA, 1984). Monoclonal antibodies can be prepared by various methods. Such methods include molecular cloning of antibodies directly from mouse hybridomas, phage displays, recombinant DNA, and primary B cells, as well as antibody discovery methods (Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al. PLOS One. 2014).

[0077] As used herein, the term “multispecific” antibody refers to an antibody having at least two binding sites, each of which has a binding affinity to an antigen epitope. As used herein, the terms “bispecific, triplicate, tetraspecific, quinticate, or hexaticate” antibody refer to an antibody having two, three, four, five, or six antigen-binding sites. For example, an antibody described herein is quinticate if it has five binding sites, and hexaticate if it has six binding sites.

[0078] The term “Guidance and Navigation Control (GNC)” refers to a multispecific protein capable of binding to at least one effector cell (e.g., immune cell) antigen and at least one target cell (e.g., tumor cell, immune cell, or microbial cell) antigen. The GNC protein may employ an antibody core structure comprising a Fab region and an Fc region having various binding domains bound to the antibody core. In this case, the GNC protein is also called a GNC antibody. The GNC protein may employ an antibody-like structure. In this case, the Fv fragment may be replaced with a non-antibody-based binding domain (e.g., NKG2D, 4-1BBL (4-1BB receptor ligand), 4-1BBL trimer for 4-1BB) or receptor.

[0079] The term "GNC antibody" refers to a GNC protein having an antibody structure capable of simultaneously binding to at least one effector cell (e.g., an immune cell) and at least one target cell (e.g., a tumor cell, immune cell, or microbial cell). As used herein, "bi-GNC, tri-GNC, tetra-GNC, penta-GNC, or hexa-GNC" antibodies refer to GNC antibodies having two, three, four, five, or six antigen-binding sites, where at least one antigen-binding site has binding affinity to immune cells and at least one antigen-binding site has binding affinity to tumor cells. In one embodiment, the GNC antibodies described herein are penta-GNC and hexa-GNC antibodies, having five and six binding sites (or binding domains), respectively. In some embodiments, the GNC antibody includes an antibody-binding domain (e.g., Fab and scFv) and does not require separate protein engineering in the Fc region. In one embodiment, the GNC antibody may include an Fc region engineered to lose effector cell function, e.g., ADCC, ADCP, CDC. Mutations include, but are not limited to, L234A / L235A / G237A / K322A and L234A / L235A / K322A (Eu numbering). In one embodiment, displacement of an Fc glycosylation site, e.g., N297A(Eu), may be used to prevent glycosylation and interfere with Fc effector function. In one embodiment, the GNC antibody used herein comprises a symmetric antibody and does not require Fc engineering to drive proper assembly of the complete protein. In contrast, many existing bispecific and multispecific antibody formats require heterodimerized Fc to combine different specificities in an asymmetric molecule. In one embodiment, the GNC antibody further has the advantage of retaining the bivalency of each target antigen. In further embodiments, the GNC antibody has the advantage of an avidity effect, resulting in high affinity and low dissociation rate for the antigen. This bivalent nature for each antigen, unlike many multispecific platforms, often loses the beneficial avidity effect that would otherwise lead to very strong antibody binding, as these multispecific platforms are monovalent for each target antigen.

[0080] The term "humanized antibody" refers to a modified antibody that has a CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. Furthermore, binding affinity can be maintained by altering the framework support residues. Methods for obtaining "humanized antibodies" are known to those skilled in the art (Queen et al., Proc. Natl Acad Sci USA, 1989; Hodgson et al., Bio / Technology, 1991). In one embodiment, a "humanized antibody" can be obtained by a genetic engineering method that can produce affinity-mature human-like polyclonal antibodies in large animals (e.g., rabbits) (USPat. No. 7, 129, 084).

[0081] The term "antigen" refers to an entity or fragment thereof that can induce an immune response in living organisms, particularly animals, and more specifically mammals, including humans. This term includes immunogens and their regions involved in antigenicity or antigenicity-determining factors.

[0082] The term "epitope," also known as "antigen determinant," refers to a part of an antigen that is recognized by the immune system, particularly antibodies, B cells, or T cells, and is a specific portion of the antigen to which an antibody binds.

[0083] The term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells to an immunogenic substance, thereby contributing to the immune response in a human or animal. The immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells to the immunogenic composition of the present invention administered to alleviate or reduce the disorder being treated.

[0084] As used herein, the term “tumor antigen” refers to antigenic molecules produced by tumor cells. Tumor antigens can elicit an immune response from the host. In one embodiment, tumor cells express tumor antigens. These tumor antigens include, but are not limited to, tumor-specific antigens (TSAs), neonatal antigens, and tumor-associated antigens (TAAs).

[0085] As used herein, the terms “specifically binding,” “specific binding,” or “specific” to a particular antigen or epitope mean a binding that is measurably different from a nonspecific interaction. Specific binding can be measured by determining the binding of a molecule by comparing it to the binding of a control molecule, which is generally a molecule of a similar structure that does not possess binding activity. Specific binding can also be determined by competition with a control molecule similar to the target. Specific binding to a particular antigen or epitope is measured at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 This can be demonstrated by antibodies having a KD of M or higher against an antigen or epitope. Here, KD refers to the dissociation rate of a particular antibody-antigen interaction. In some embodiments, multispecific antibodies that specifically bind to an antigen have a KD of 20, 50, 100, 500, 1000, 5,000, or 10,000 times or more against the antigen or epitope compared to a control molecule. Furthermore, specific binding to a particular antigen or epitope can be demonstrated by antibodies whose KA or Ka against the antigen or epitope is at least 20, 50, 100, 500, 1000, 5,000, or 10,000 times or more than that of the control. KA or Ka refers to the association rate of a particular antibody-antigen interaction.

[0086] The term "staple" means that two domains are covalently bonded. In one embodiment, the two domains may be covalently bonded via at least one disulfide bond. For example, an scFv domain having at least one disulfide bond connecting VH and VL is called a staple scFv. A Fab region having at least one disulfide bond connecting a light chain portion and a heavy chain is called a staple Fab.

[0087] Examples The following examples are for illustrative purposes only and do not limit the present invention. Those skilled in the art will readily recognize a variety of non-essential parameters that can be changed or modified to produce essentially the same or similar results.

[0088] Example 1: Stability of staple-binding domain and penta and hexa-GNC antibodies In multispecific GNC antibodies such as tetra-GNC antibodies, the heavy chain contains three scFv domains and a Fab region to constitute quadruple binding specificity, while the light chain may remain unmodified. In penta-GNC antibodies, one scFv domain is added to either the N-terminus or C-terminus of the light chain to obtain quintuple binding specificity (Figure 1 and Table 1). When scFv domains are bound to both the N-terminus and C-terminus of the light chain, the antibody acquires fifth and sixth binding specificities, becoming a hexa-GNC antibody. Modification of both the heavy and light chains may affect the stability of the antibody. To maintain the stability and independence of all binding domains in either penta or hexa-GNC antibodies, each Fv fragment and scFv domain is modified with V L 100 and V H One method involves introducing a disulfide bond at 44 (Kabat), i.e., stapling it. L and V H The disulfide bond between them can be applied to all scFv domains to stabilize the entire structure. Alternatively, the disulfide bond may be introduced at any position in at least one selected scFv domain.

[0089] To analyze the effect of the staple scFv domain, a pair of penta-GNC antibodies (SI-1P1 and SI-1P2) with identical binding specificity (sequence numbers 1-4 and 5-8, respectively) were prepared. Based on the numbering system in Figure 1, the heavy chains of these two antibodies were αCD3 scFv at D1 and αEGFR V at D2. H (Fv-CH1-Fc configuration), with αPD-L1 at D3 and α4-1BB at D4, and the light chain is αEGFR V at D5. L It also has αHER3 scFv. SI-1P2 (not SI-1P1) has a "staple" scFv domain in D1, D3, D4 and D4, i.e., as shown in Table 1, its heavy chain D1 has αCD3 scFv[V H 44G->CV L 100G->C], αPD-L1 scFv[V in D3 H 44G->CV L 100G->C], α4-1BB scFv[V on D4 H 44G->CV L It has 100G->C] and its light chain contains αHER3 scFv[V H 44G->CV L It has 100G->C] (Kabat numbering).

[0090] Both SI-1P1 and SI-1P2 were cloned into vector pTT5 according to a modular cloning strategy using restriction sites HindIII / SalI / NheI / BamHI / BspEI / PacI. These pentaGNC antibody constructs were expressed at acceptable titers for 5 and 9 days, respectively, using both HEK and ExpiCHO expression systems. After purification using a 5 mL MabSelect Protein A column, size exclusion was performed using a hiload 16 / 600 200 pg preparative SEC column on either an Akta Avant or Purifier system. SEC aggregation was analyzed using Waters HPLC connected to a multi-angle light scattering detector (MALS, Wyatt systems), and the exact molecular weight was determined by the dn / dc calculation method. In the next analysis, the melting temperature of the obtained proteins was determined using a dynamic light scattering instrument (Wyatt systems). As can be seen from all analyses performed, as shown in Table 2, disulfide-bonded (i.e., "stapled") pentaGNC antibodies exhibit higher stability.

[0091] Antibody proteins are almost always purified by protein A affinity chromatography. Protein A resin is the C of the Fc domain. H2 -C H3 It captures the antibody at the binding site at the interface. However, protein A is V H3 Family Fv's V H It also joins to the domain. V H This is not a problem for most antibody-based platforms because the domain is usually located in the heavy chain. However, V H3 When scFv containing V is attached to the light chain, H The domain can bind to the protein A resin during purification. This contaminates the heavy-chain-light-chain heterotetramer with light-chain monomers and dimers. Therefore, the light chain can become V H3 A potential obstacle in producing multispecific antibodies containing domains is the presence of additional contaminants in the protein A eluate. This is particularly problematic when the light chain is expressed more efficiently than the heavy chain, and large amounts of light chain contaminants must be purified along with the desired protein assembly.

[0092] To effectively disrupt the protein A binding of VH3 family members, a structural approach was employed to block the binding interface. Crystal structure 1DEE (Graille M. et al. Proc. Nat. Acad. Sci. 2000.) is V H3 This shows that residue R19 (Kabat numbering) in this molecule directly contacts both side chains of protein A domain D. In particular, the interaction can be significantly weakened by releasing contact with Q32 and D36. Therefore, R19 was mutated to serine. This is because the serine side chain is short and does not form these interactions. Furthermore, since S19 is naturally present in other VH family members, it suggests that it may be less immunogenic than other substitutions.

[0093] The mutation R19S (Kabat numbering) is present on the GNC light chain. H3 The R19S mutation was incorporated into the FR1 region of the VH domain for the contained scFv. Specifically, the penta-GNC antibody SI-77P1 (SEQ ID NO: 41-44) has an R19S mutation in the light chain sequence encoding anti-CD19 scFv in domain 6, while the hexa-GNC antibodies SI-55-H11 (SEQ ID NO: 53-56), SI-55H12 (SEQ ID NO: 57-60), SI-77H4 (SEQ ID NO: 61-64), and SI-77H5 (SEQ ID NO: 65-68) have an R19S mutation in the anti-HER3 scFv domain in D5 and the light chain sequence encoding anti-CD19 scFv in domain 6. Since the target residue is located at the protein A binding interface (4), the R-to-S mutation interferes with the interaction with protein A. By removing the binding of protein A to the light chain scFv, it is possible to prevent light chain monomers and dimers from binding to protein A during purification. This results in a more homogeneous product free from light chain contaminants. Up to 2 Vs per light chain H3 In the case of hexa-GNCs that may contain scFv, this mutation is very important because it allows for the efficient purification of the target product.

[0094] Wild-type IgG1 antibodies contain an active Fc domain that binds to the Fcγ receptor on immune cells and C1q, the first component of the complement cascade. This binding ability allows antibodies with active Fc to induce effector functions (including antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity against antigen-carrying cells). However, in the context of T-cell redirection, the active Fc domain can exacerbate cytokine release syndrome and cause off-target cytotoxicity (Strohl & Naso, Antibodies, 2019). Therefore, a null FC domain incorporating silencing mutations that weaken binding to the Fcγ receptor and complement can enhance the effectiveness of T-cell redirection antibodies by reducing cytokine release syndrome and increasing tumor infiltration (Wang et al., Cancer Immunol. Res. 2019). Many point mutations have been introduced to weaken the interaction with the Fcγ receptor or C1q and reduce Fc effector function (Saunders, Frontiers Immunol. 2019). Among these, the L234A, L235A, and G237A mutations reduce ADCC and ADCP by decreasing binding to the Fcγ receptor. The K322A mutation reduces binding to C1q and removes CDC. Furthermore, the N297A mutation removes the Fc glycosylation site, generating a non-glycosylated Fc domain that does not interact as strongly with the receptor.

[0095] To create therapies that reduce the risk of cytokine storms and improve the quality of tumor penetration by reducing peripheral Fcγ receptor binding, we incorporated Fc silencing mutations into the GNC platform. Examples of molecules include different null Fc versions, demonstrating that a range of Fc archetypes can be used on the GNC platform. Therefore, mutations used to modulate the effector function of monoclonal and multispecific antibodies can also be efficiently incorporated into the GNC platform.

[0096] Example 2: Optimization of binding domains The usefulness of multispecific GNC antibodies is determined by selecting binding specificity, while the effectiveness of antibodies can be improved by optimizing commonly used binding domains. Penta-GNC and hexa-GNC antibodies (collectively referred to as GNC antibodies, as shown in Table 1) were cloned, expressed, and produced using the same materials and methods as those used to produce the SI-1P1 and SI-1P2 antibodies described in Example 1.

[0097] The anti-CD3 variable domain sequence 284A10 (applicant's application number PCT / US2018 / 039143; the entire sequence is incorporated herein) is used in scFv(V H -V L or V L -V H Unmodified, stapled (284A10 staple, SEQ ID NOs. 89-92), humanized (284A10 H1, SEQ ID NOs. 93-96), or humanized and stapled (284A10 H1 staple, SEQ ID NOs. 97-100) sequences encoding either the orientation or Fab of the heavy chain monomer were used. Another anti-CD3 variable domain sequence 283E3 was identified, cloned, and humanized as 283E3 H1 (SEQ ID NOs. 101-104) used to encode the Fab region at D2 of each heavy chain monomer in seven penta-GNC antibodies (Table 1).

[0098] The anti-PD-L1 variable domain sequence PL221G5 (applicant's application number PCT / US2018 / 039144; the entire sequence is incorporated herein) was humanized and used to encode either the "non-staple" or "staple" (SEQ ID NO: 105-108) scFv domain located at D3 of the heavy chain monomer.

[0099] The anti-4-1BB variable domain sequence 466F6 (applicant's application number PCT / US2018 / 039155; the entire sequence is incorporated herein) was humanized and used to encode either the "non-staple" or "staple" (SEQ ID NO: 109-112) scFv domain at D4 of the heavy chain monomer.

[0100] Example 3: Octet analysis of binding affinity To evaluate the functionality of the GNC antibody, the binding affinity of individual domains of the penta-GNC antibody was assessed using Biolayer Interferometry (Octet384 system). The penta-GNC antibody was captured on a probe using anti-human Fc (AHC chip), and individual epitopes (CD3ε / δ) and EGFR were prepared. 4-1BB and PD-L1 (Acro Biosystems) were used as analytes, and kinetic analysis (K) was performed. off / K on ) due to the dissociation constant (K D The following was determined. As shown in Table 3, the binding constants for any individual domain of SI-1P1 or SI-1P2 were all within the reported range, and the affinity of the monoclonal antibody or scFv molecule alone was determined in advance.

[0101] Octet analysis confirmed that the GNC antibody maintained binding to all of its allogeneic antigens. The GNC antibody was loaded into an AHC sensor at 10 ug / ml for 180 seconds. This was followed by a 60-second baseline step, a 180-second association step with 100 nM commercial human antigen, and a 360-second dissociation step. Throughout all steps, the sample was in Octet buffer (PBS containing 0.1% Tween 20 and 1% BSA). A 1:1 binding model was used to perform a fit and extract the affinity KD values ​​reported in Table 4. The data demonstrate that each binding domain retains its binding affinity even when positioned at different locations on the GNC antibody.

[0102] Example 4: T cell activation To validate the functionality of GNC antibodies, T cell activation of penta-GNC antibodies was evaluated. T cell activation assays were used to compare the efficacy of SI-1P1, which binds to both EGFR and HER3, with that of EGFR-tetra-GNC antibody (which binds to EGFR but not HER3), FITC-quadruplicate antibody (which does not bind to either EGFR or HER3), and bispecific antibody (which binds only to EGFR and HER3). Human pancreatic cancer cells expressing high levels of EGFR and low levels of HER3 (BxPC3) were used as target cells (Table 5). After treating BxPC3 cells with a dissociation reagent (TrypLE Express), quadruplicates were inoculated into 384-well plates at a density of 1500 cells / well using BioTek EL406 and allowed to adhere for 24 hours. Subsequently, Jurkat CD3 NFAT effector cells (Jurkat Lucia cells, Invivogen) were added in a 5:1 cell ratio, and GNC antibody was added in 10-point, 10-fold serial dilutions from 50 nM to 0.5 fM. The cells were incubated for 4 hours. Promega Bright-Glo reagent was added, and the data was read out. Luminescence was measured using a Clariostar Plus microplate reader (BMG-Labtech). The data were plotted on a logarithmic scale using Graphpad prism and fitted to a nonlinear variable gradient equation (Figure 2). The data show that the penta-GNC antibody (SI-1P1) exhibits similar efficacy (SI-1P1=1.456pM, EGFR-tetra-GNC=1.028pM, FITC-tetra-GNC=13.41pM) and higher maximal T cell activation (SI-1P1=6464, EGFR-tetra-GNC=5161, FITC-tetra-GNC=2835) compared to the control antibodies (EGFR-tetra-GNC antibody, FITC-tetra-GNC antibody, and control bispecific antibody) (Table 6).

[0103] Example 5: T cell-dependent cytotoxicity (TDCC) TDCC is a standard feature of antibody therapy for treating cancer and other diseases. To evaluate GNC antibody-mediated TDCC, SI-1P1 (a penta-GNC antibody capable of binding to the tumor antigens EGFR and HER3) was used and compared with a control antibody. The control antibodies included an EGFR-tetra-GNC antibody that binds only to EGFR, a FITC-quadrispecific antibody that does not bind to either EGFR or HER3, and a control bispecific antibody that binds only to the tumor antigens EGFR and HER3 (Table 6). Serially diluted antibodies (0 to 30 nM; dilution ratios 1 to 5) were added to a total volume of 50 µl in a white 384-well plate containing luciferase-treated (luciferized) MDA-MB-231 or HeLa cells (both with high EGFR and low HER3 (Table 5); inoculated 24 hours prior and grown at 37°C) and activated T cells (inoculated immediately prior to drug administration; effector:target = 5:1). After another 72 hours, 20 µl of Bright-Glo (Promega) was added to the wells, and the luminescence relative to the viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. The data were fitted to a sigmoid function to calculate the EC50 values ​​(Figure 3). The MDA-MB-231 cells shown in Figure 3A had EC50 values ​​of 0.01575 pM (SI-1P1), 0.01646 pM (EGFR-tetra-GNC control antibody), and 1.882 pM (FITC-quadrispecific control antibody), while the cells shown in Figure 3B had EC50 values ​​of 1.161 pM (SI-1P1), 1.635 pM (EGFR-tetra-GNC control antibody), 3736200 pM (FITC-quadrispecific antibody), and 4500 pM (bispecific control antibody). The data shows that as the number of binding specificities increases, the penta-GNC antibody exhibits higher TDCC efficacy compared to a control antibody with fewer binding specificities.

[0104] Example 6: NKG2D receptor as the binding domain By increasing the number of binding specificities, the GNC antibody binds not only to T cells, but also to subsets of T cells, natural killer cells and other types of immune cells. On the other hand, additional binding specificities can substitute for cellular response or recognition against target cells. For example, NKG2D is a major recognition receptor for detecting and eliminating transformed cells and infected cells because its ligand is induced during cellular stress, or as a result of genomic stress such as viral infection or cancer. In humans, NKG2D is expressed on NK cells, T cells and CD8 + T cells. In NK cells, NKG2D functions as an activating receptor and can itself trigger cytotoxicity. CD8 + In T cells, the function of NKG2D is to transmit co-stimulatory signals to activate the T cells. By adding NKG2D as a binding specificity for the GNC antibody, the cytotoxicity and efficacy of the antibody as a single multifunctional therapeutic agent can be improved. As used herein, the penta GNC antibodies SI-49P1 and SI-49P3 (SEQ ID NOs: 17-20 and SEQ ID NOs: 21-24, respectively) were generated by adding the NKG2D receptor as a binding domain to D5 (Table 1). The affinity of the NKG2D moiety of SI-49P3 for human MICA (Table 4) was found to be within the expected range, demonstrating that NKG2D can function as a receptor for the penta GNC antibody to bind its ligand.

[0105] Both SI-49P3 and SI-49P1 can recognize a single tumor antigen via their Fab region and extend their multi-binding specificity to CD3, PD-L1, 4-1BB, and NKG2D. To demonstrate that SI-49P3 retains its ability to activate T cells, BxPC3 cells were treated with a dissociation reagent (TrypLE Express), then inoculated into 384-well plates at a density of 1500 cells / well using a BioTek EL406 in a quadruplicate and incubated for 24 hours. Subsequently, Jurkat CD3 NFAT effector cells (Jurkat Lucia cells, Invivogen) were added in a 5:1 cell ratio, and GNC reagent was added in 10-point 10-fold serial dilutions from 50 nM to 0.5 fM, followed by incubation for 4 hours. Promega Bright-Glo reagent was added and readout was performed, and luminescence was measured using a Clariostar Plus microplate reader (BMG-Labtech). The data were plotted on a logarithmic scale using Graphpad prism and fitted to a nonlinear variable gradient equation (Figure 4A). The data show that the addition of the NKG2D receptor did not affect T cell activation, and that the penta-GNC antibody can induce effective T cell activation (EC50 = 88.1 pM) and can target tumor cells by involving T cell antigens.

[0106] TDCC of the NKG2D class of penta-GNC antibodies was evaluated using SI-49P1. Control antibodies included tri-GNC antibodies lacking binding specificity to both PD-L1 and 4-1BB, and tetra-GNC antibodies lacking the NKG2D receptor. Serially diluted (0 to 30 nM; dilution ratios 1 to 5) GNC proteins were added to 384 white wells containing luciferase-treated MDA-MB-231 cells (MICA and mesothelin expressing; inoculated 24 hours prior and grown at 37°C) and activated T cells (inoculated immediately prior to drug administration; effector:target = 5:1) in a total volume of 50 μl. After a further 72 hours, 20 μl of Bright-Glo (Promega) was added to the wells, and luminescence relative to luciferase-treated tumor cell viability was determined using a Clariostar plate reader. The EC50 values ​​were calculated by fitting the data to a sigmoid function, resulting in values ​​of 0.1865 pM (SI-49P1), 0.3433 pM (tri-GNC control antibody), and 5.356 pM (tetra-GNC control antibody) (Figure 4B). The data indicate that adding the NKG2D receptor to the tetra-GNC antibody improves the efficacy of TDCC, and that adding the αPD-L1 and α4-1BB domains to the tri-GNC antibody, which has binding specificity to NKG2D, significantly improves the efficacy of TDCC. In other words, GNC antibodies can modulate, coordinate, and induce an optimized immune response against target cells (e.g., cancer) by adapting to multiple binding specificities.

[0107] Example 7: NKG2D receptor located at D2 of antibody-like GNC protein To test the efficacy of an antibody-like GNC protein with a native receptor in D2, the sequence for human NKG2D (residues F78 to V216) was cloned and placed in the context of an expression plasmid encoding SI-49P10 (αCD3xNKG2DxαPD-L1xα4-1BBxαEGFR, SEQ ID NOs. 117-200) in D2. H and V LDomain substitution was performed. Following the above materials and methods, SI-49P10 was expressed and protein A was purified, resulting in an antibody-like GNC protein with very low aggregation (analysis SEC: target peak 95.64%) and a non-antibody binding moiety (e.g., NKG2D receptor) on the D2 of the heavy chain, indicating a high probability of high stability. Penta-GNC proteins SI-49P6 (αCD3xNKG2DxαPD-L1xα4-1BBxαCD19, SEQ ID NOs. 123-126) and SI-49P7 (αCD3xNKG2DxαPD-L1x41BBL trimer xαCD19, SEQ ID NOs. 127-130) were cloned, expressed, and purified in the same manner as above.

[0108] To ensure that the NKG2D dimer retains its full functionality, Octet binding to human MICA was evaluated. SI-49P10 was loaded onto an AHC chip and bound to His-tagged MICA. Extracted KD values ​​confirmed that NKG2D retains binding activity when it is located at the D2 position (Table 4). SI-49P10 is K D The value was 1.84 nM. For comparison, SI-49P3 (NKG2D dimer in D5) is a similar K D It had a value of 1.39 nM. Other domains of SI-49P10 also maintained high binding affinity to their alloantigens (Table 4). Similarly, the binding of SI-49P6 and SI-49P7 to MICA was determined by loading biotinylated human MICA onto an SA chip and observing the binding to serial dilutions (0 to 100 nM) of the GNC protein used as the analyte. D The values ​​were 7.763 nM (SI-49P6) and 10.67 nM (SI-49P7), further demonstrating that the receptor protein at position D2 retains target binding (Table 4). These K proteins use the antigen as a loaded ligand. DThe value shows a slightly lower affinity compared to experiments loaded with GNC protein, which may be attributed to the inactive conformation or incompletely exposed epitope of MICA protein when loaded as a ligand. Nevertheless, the potent femtomolar (<1 pM) TDCC against MICA-carrying MDA-MB-231 cells induced by these proteins with NKG2D at position D2 (Figure 8) indicates that the NKG2D receptor retains active binding at position D2. This demonstrates that the GNC platform has extremely high adaptability to the arrangement position of each domain.

[0109] Example 8: 4-1BB ligand as binding domain 4-1BB is a co-stimulatory immune checkpoint TNFR receptor expressed by activated T cells and NK cells. 4-1BB ligand or CD8 +Activation of T cells by agonist antibodies leads to increased proliferation, cytokine production, and survival. To optimize the 4-1BB-mediated immune response, the functionality of different domains was evaluated using a 4-1BB activation reporter bioassay. The 4-1BB activation assay is based on the method of the Promega 4-1BB Bioassay Kit (SKU: JA2351). This assay consists of a genetically modified Jurkat T cell line expressing human 4-1BB and a luciferase reporter (called a 4-1BB effector cell) driven by a response element responsive to 4-1BB ligand / agonist antibody stimulation. The 4-1BB effector cells are cultured in RPMI-1640 containing 10% FBS. Prior to this assay, these cells are counted and re-seeded at 500 cells / well in 384 wells (Corning 3570). 96-well dilution blocks were stamped into the 384-well quadrant using a robot (Opentrons OT-2 liquid processing robot), and the test substance experiment was performed using quadruple replicas. The 4-1BB assay plate was incubated for 6 hours. The 4-1BB activation curve was read using the Promega Bright-Glo luciferase assay kit. In short, 20 μL was added to the 4-1BB assay plate and incubated for approximately 15 minutes, after which the resulting luminescence was measured using a BMG Clariostar plate reader. The activation curve was analyzed and constructed using GraphPad software with 4PL curves (Figure 5). As can be seen from the results, the 4-1BB ligand trimer (4-1BBL trimer, SEQ ID NOs. 113-114) induces strong activation of 4-1BB signaling compared to the monomer 4-1BB ligand, monomer Fc, and anti-4-1BB scFv. PentaGNC antibodies SI-55P4, SI-55P10, and SI-79P3 (SEQ ID NOs. 29-32, 37-40, and 49-52, respectively) were prepared to have the 4-1BBL trimer as a binding domain at D4 (Table 1 and Figure 7).

[0110] The bioactivity of the GNC protein with NKG2D at the D2 position was confirmed using MICA-supported MDA-MB-231 target cells via a TDCC assay (Figure 8). The target cell:effector cell ratio was 1:5, and the assay was performed for 72 hours after adding the drug dilution and T cells to tumor cells. The resulting EC50 values ​​were very effective (SI-49P6, 0.7366 pM and SI-49P7, 0.1094), confirming the ability of NKG2D to target T cells and kill tumor cells. Therefore, by placing the receptor NKG2D as the binding domain at the D2 position of GNC, a stable GNC protein that induces potent TDCC can be obtained.

[0111] Example 9: Humanized EGFR-binding domain Cetuximab is a chimeric mouse / human monoclonal antibody for the treatment of EGFR-expressing metastatic colorectal cancer, non-small cell lung cancer, and head and neck cancer. Humanized antibodies are obtained. In this example, humanized sequences encoding anti-EGFR binding (H1, H4, H7, and H7-staples) (sequence numbers 69-72, 73-76, 77-80, and 81-84, respectively) were cloned into expression cassettes producing anti-EGFR(D2) pentaGNC antibodies (SI-77P1) and anti-EGFR(D1) pentaGNC antibodies (SI-55P3, SI-55P4, SI-79P2, SI-79P3, and SI-55P9, respectively) (sequence numbers 25-28, 29-32, 45-48, 49-52, and 33-36, respectively) (Table 7). Each expression cassette was transfected into 25 mL of ExpiCHO and expressed for 8 days. The respective pentaGNC antibodies were then collected and purified by protein A affinity chromatography. Antibodies with good titers were obtained (Table 7). Analytical SEC data after protein A purification demonstrated that pentaGNC antibodies with humanized anti-EGFR domains could be expressed with low aggregation (Table 7). Using Octet, the ability of pentaGNC antibodies containing humanized anti-EGFR domains H1, H4, or H7 to bind to human EGFR was verified (Figure 6 and Table 7). Each pentaGNC antibody was loaded at 10 μg / ml using an AHC sensor and bound to serial dilutions (maximum 200 nM, 1:2.5 dilution) or to a single 100 nM concentration of His-tagged human EGFR. Global fitting to the resulting 1:1 binding model demonstrated that pentaGNC antibodies bind to EGFR with low nanomolar affinity (Table 7).

[0112] To produce hexa-GNC antibodies, the humanized anti-EGFR domain H7 (SEQ ID NO: 77-80) was cloned into an expression cassette for producing anti-EGFR hexa-GNC antibodies. The humanized binding domain was placed in Fab or scFv at D1 in hexa-GNC antibodies SI-77H4 (SEQ ID NO: 61-64) and SI-55H11 (SEQ ID NO: 53-56). The control antibody SI-77H5 (SEQ ID NO: 65-68) contains the anti-EGFR binding Fab region encoded by cetuximab mice. The expression cassettes were transfected into 25 mL of ExpiCHO and expressed for 8 days. After that, each hexa-GNC antibody was collected and purified by protein A affinity chromatography. Hexa-GNC antibodies with good titers were obtained (Table 8). Analytical SEC data after protein A purification demonstrated that hexa-GNC antibodies containing the humanized anti-EGFR domain could be expressed with low aggregation (Table 8). Using Octet, we verified that these hexa-GNC antibodies containing humanized anti-EGFR domains could each bind to human EGFR (Tables 4 and 8). The hexa-GNC antibodies were loaded at 10 μg / ml using an AHC sensor and bound to His-tagged human EGFR at serial dilutions (up to 200 nM, 1:2.5 dilution) or a single 100 nM concentration. Global fitting to the resulting 1:1 binding model demonstrated that each hexa-GNC antibody bound to EGFR with low nanomolar affinity (Table 8).

[0113] Example 10: Humanized CD19-binding domain CD19 is a biomarker for B lymphocyte development and the diagnosis of lymphoma. CD19-targeted therapy based on T cells expressing CD19-specific chimeric antigen receptors (CAR-T) is effective due to their antitumor capabilities. + It is used in patients with lymphoma and leukemia (e.g., non-Hodgkin lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia). In this specification, a humanized CD19 binding domain is preferred.

[0114] All computational steps were performed using the Discovery Studio package (Dassault Systemes). First, a structural model was constructed using the mouse BU12 sequence. A Hidden Markov Model (HMM) was used to identify the antibody framework region in the input sequence and align it with a database of antibody variable domains. The model was then constructed and cored using MODELLER software based on this alignment. CDR loop modeling was performed by structural mapping of the CDRL1, CDRL2, CDRL3, CDRH1, and CDRH2 regions to known normative classes, and the loop model was constructed similarly to the framework. The framework region derived from the mouse BU12 antibody was aligned and matched to the nearest human germline sequence. The CDR region was copied to the human sequence, excluding important structural residues (Vernier residues [Almagro and Fransson, 2008]). Mutations predicted to stabilize previously constructed structural models were calculated and evaluated using the steepest descent method with a RMS gradient tolerance of 3 and 1000 steps. Then, conjugation gradient minimization was performed to select stabilizing mutations that matched frequent human residues based on the individual and combined -ΔΔG pair initial models. Humanization of the resulting final sequences was tested using the Abysis web server based on the method of Abhinandan and Martin (2007).

[0115] To confer binding specificity to CD19 targeting B-cell malignancies to penta- or hexa-GNC antibodies, the sequences encoding anti-CD19V L and V H The sequence that codes for the domain, modified V L Sequence IDs 87, 88, 121, 122, 131, 132 and modified V have the following characteristics: L and R19S mutation-containing V HWe selected from sequence numbers 85, 86, 87, and 88 (R19S mutations in Table 1 and Example 1) and bound them to a (G4S)x4 linker to form an anti-CD19scFv domain. The corresponding gene sequences were cloned to different positions in penta- or hexa-GNC antibodies and digested into pTT5 expression plasmids using restriction enzymes to obtain the appropriate heavy or light chain portion. Anti-CD19 penta-GNC and hexa-GNA antibodies were prepared and characterized as shown in Table 1. Octet analysis of CD19 binding affinity showed that each GNC antibody retained CD19 binding affinity within the expected range when placed on the light chain monomer of the GNC antibody (Table 4).

[0116] Example 11: PentaGNC antibody having an optimized binding domain PentaGNC antibodies were evaluated in a TDCC assay by optimized specific binding for EGFR, CD19, and 4-1BB receptors. SI-55P9 and SI-55P10 (SEQ ID NOs. 33-36 and 37-40, respectively) are a pair of pentaGNC antibodies with identical binding specificity. SI-55P9 possesses a humanized anti-EGFR binding domain, while SI-55P10 activates 4-1BB signaling in the anti-4-1BB binding domain of SI-55P9 using a 4-1BBBL trimer. To evaluate the impact of these differences in the TDCC assay, serial dilutions (0 to 30 nM; 1 to 5 dilution ratios) of pentaGNC proteins SI-1P1, SI-55P9, and SI-55P10 were added to a 384-well white plate containing luciferase-treated BxPC3 (high EGFR expression) cells (inoculated 24 hours prior and grown at 37°C) and activated T cells (inoculated immediately prior to drug administration; effector:target = 5:1 (SI-1P1), 7:1 (SI-55P9 and SI-55P10)) in a total volume of 50 μl. After a further 72 hours, 20 μl of Bright-Glo (Promega) was added to the wells, and luminescence relative to the viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. As shown in Figure 7, the potency of SI-55P9 and SI-55P10 was similar to that of SI-1P1. The EC50 values ​​calculated by fitting the data to a sigmoid function were 0.2814 pM (SI-1P1), 0.4871 pM (SI-55P9), and 0.7358 pM (SI-55P10), all within the picomolar range. As can be seen from these results, the composition of binding specificity determines the efficacy of the penta-GNC antibody in the TDCC assay, and the optimization of each binding domain can be more appropriately evaluated by other in vitro and in vivo assays.

[0117] Example 12: Cytotoxicity of redirected T cells Any antibody-based binding domain can be converted to Fab or scFv format and directly inserted into a GNC antibody. For example, a GNC antibody is characterized by the addition of a fifth and / or sixth binding domain to its light chain portion. When binding specificity on the heavy chain can be dedicated to targets such as CD3, PD-L1, and 4-1BB, which are frequently used, the utility of the GNC platform can be flexible in that it allows for the selection of target tumor antigens and the pairing of less flexible heavy chains with desired light chain portions. In this specification, three tetra-GNC antibodies were selected (Applicant's application number PCT / US2019 / 024105; the entire antibody is incorporated herein) and evaluated using in vitro redirected T-cell cytotoxicity (RTCC) assays and in vivo human tumor xenograft models.

[0118] SI-35E20 is a tetra-GNC antibody capable of binding to 4-1BB (D1), PD-L1 (D2), ROR1 (D3), and CD3 (D4) (Table 1). The ability of SI-35E20 to induce RTCC over 4 days was determined by live-cell imaging of cultures containing PBMCs (single donor) and red fluorescently labeled tumor cells. NucRed-transduced A549 lung adenocarcinoma cells were targeted using PBMCs (50,000 cells / mL) in a 4:1 (PBMC:A549) ratio. Assay wells were triple-repeated with 1 nM SI-35E20 or GNC-free (buffer only) as negative controls, and target cell proliferation was monitored for 94 hours. These data demonstrate that SI-35E20 can inhibit the proliferation of target cancer cells over time (Figure 9).

[0119] SI-38E17 is a tetra-GNC antibody capable of binding to CD3(D1), CD19(D2), PD-L1(D3), and 4-1BB(D4) (Table 1). The effect of SI-38E17-mediated RTCC on cancer cells was evaluated using Nalm-6Nuc-GFP (human leukemia cell line) as the target cell line and PBMCs from a single donor as effector cells. The RTCC assay was performed with an E:T ratio of 1. Target cell proliferation was detected by tracking SI-38E17-mediated RTCC over 48 hours using IncuCyte at 100 pM. SI-38E17 mediated potent RTCC functional activity against Nalm-6 (Figure 10). The obtained data demonstrate that tetra-GNC antibodies, such as SI-38E17, can inhibit the proliferation of target cancer cells over time.

[0120] SI-39E18 is a tetra-GNC antibody capable of binding to CD3, EGFRvIII, PD-L1, and 4-1BB. The RTCC assay confirmed greater cell killing than the solvent control, as shown in Figure 11. Figure 11 shows the average of time-course red fluorescence intensity measurements for three different PBMC donors. Measured at fluorescence intensity during the first 24 hours of culture, the number of target cells increased in the presence of SI-39E18 or the buffer control. Here, effector cells were pre-incubated with SI-39E18 or the control for 3 days before adding the target cells. Between 24 and 48 hours after adding the target cells to the culture, the number of target cells began to decrease in the presence of SI-39E18 stimulation, while it did not decrease in the wells containing the buffer control. The more moderate decrease in target cells in the buffer control samples under both conditions after 48 hours is likely due to nutrient depletion or an allogeneic T cell response to MHC mismatch target cells. The data obtained demonstrates that tetra-GNC antibodies, such as SI-39E18, can prevent the proliferation of target cells over time.

[0121] Example 13: Human tumor xenograft model In a mouse xenograft model, the ability of SI-38E17 to slow in vivo tumor growth was investigated (Figure 12). Human B-cell leukemia cells (JVM-3) were transplanted in 5 × 10⁶ units. 6 Cells / mouse were subcutaneously transplanted into the right flank of NCG mice, and the tumor volume was 50-80 mm. 3 When it reaches 2 × 10 7 Cells / mice were administered by intraperitoneal injection. Each group consisted of 5 animals, and the labeled dose was administered intravenously once daily. The tumor volume after SI-38E17 administration is shown in the figure. On day 16, the tumor volume in the solvent group was 1298 mm³. 3 The results showed that all three doses of SI-38E17 exhibited significant tumor suppression effects, with the intermediate dose of 0.005 mg (drug:TCR=12.5:1) showing the best tumor suppression (TGI=84%). While all mice in the solvent control group died before day 22, tumors were eliminated before day 40 in one mouse in the low-dose group, three in the intermediate-dose group, and one in the high-dose group. Therefore, tetra-GNC antibodies, such as SI-38E17, exhibit strong tumor suppression effects in vivo across multiple doses.

[0122] The ability of SI-39E18 to slow tumor growth in vivo was investigated in a mouse xenograft model (Figure 13). 5 × 10¹⁶ NCG mice were implanted in the right flank. 6 UM-UC-3-EGFR VIII cells derived from individual human bladder cancers were inoculated. The average tumor volume was 50-80 mm². 3 When it reaches 5 × 10 6Human PBMCs (100 µl) in mice were intraperitoneally injected, and different doses of SI-39E18 were administered intravenously. Each group consisted of 5 animals, and the indicated dose was administered intravenously once daily for a total of 18 doses. Day 1 of administration was defined as D1. Tumor growth after SI-39E18 administration is shown in the figure, demonstrating that SI-39E18 induces strong suppression of tumor growth across multiple doses. On the day of discontinuation (D18), tumor volume remained at 0 for 3 consecutive days in all dose groups (low dose group 0.001 mg, intermediate dose group 0.01 mg, high dose group 0.1 mg), while volume significantly increased in the solvent group. Therefore, tetra-GNC antibodies, such as SI-39E18, exhibit strong in vivo biological activity across multiple doses.

[0123] Example 14: Exhibition of more complete cytotoxicity We prepared hexa-GNC antibodies as monoantibody therapeutics and investigated their multifunctionality. SI-55H11 (SEQ ID NOs. 53-56) is a hexa-GNC antibody with binding specificity to CD3 (D1), EGFR (D2), PD-L1 (D3), and 4-1BB (D4) on the heavy chain monomer, and to HER3 (D5) and CD19 (D6) on the light chain monomer (Table 1). By TDCC assay, we determined the effect of targeting PD-L1 and 4-1BB on T cell-mediated tumor cell killing compared to triplicate antibodies targeting both CD3 (D1) on T cells and EGFR (D2) and HER3 (D5) on tumor cells (Table 4). Serially diluted (0 to 30 nM; dilution ratio 1 to 5) bird or hexa-GNC antibodies were added to a 384-well white plate containing luciferase-treated BxPC3 (high EGFR expression) cells (inoculated 24 hours prior and grown at 37°C) and activated T cells (inoculated immediately prior to drug administration; effector:target = 5:1) in a total volume of 50 μl. After a further 72 hours, 20 μl of Bright-Glo (Promega) was added to the wells, and luminescence relative to the viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. The data were fitted to a sigmoid function to calculate EC50 values ​​and maximum virulence (Figure 14). The data show that the hexa-GNC antibody exhibits more complete virulence compared to the trispecific antibody (bottom plateau = 46.92% viability; SI-55H11 bottom plateau = 2.992% viability). The data suggests that activating T cells along with CD3, while simultaneously targeting immunomodulatory proteins such as the PD-L1 immune checkpoint, and activating 4-1BB is an effective combined strategy for inducing a GNC response of the immune system against target cells, leading to more complete tumor depletion.

[0124] Example 15: Targeting of multiple tumor antigens As shown in Examples 12 and 13, attempts can be made to immobilize immune targets on the heavy chain. The binding domain on the light chain portion can be dedicated to tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), and nascent antigens. In this specification, several hexa-GNC antibodies were prepared and subjected to TDCC assays. The GNC antibodies were evaluated to determine whether additional tumor target specificity could increase T cell-mediated killing of tumor cells (Figure 15). In this specification, SI-55P9 (SEQ ID NO: 33-36), a penta-GNC antibody that can bind to EGFR, CD3, PD-L1, and 4-1BB via the heavy chain monomer and to CD19 via the light chain monomer, was compared with SI-55H11 (SEQ ID NO: 53-56), a hexa-GNC antibody that has the same binding specificity but adds a sixth specificity to HER3 via the light chain monomer. Each GNC antibody, serially diluted (0 to 30 nM; dilution ratio 1 to 5), was added to a 384-well white plate containing 500 luciferase-treated BxPC3 (high EGFR; low HER3) cells (inoculated 24 hours prior and grown at 37°C) and 2500 activated T cells (inoculated immediately before drug administration; effector:target = 5:1) in a total volume of 50 μl. After a further 72 hours, 20 μl of Bright-Glo (Promega) was added to the wells, and luminescence relative to the viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. The data were fitted to a sigmoid function to calculate EC50 values. The data indicate that the hexa-GNC antibody exhibits higher potency in the TDCC assay (SI-55P9 EC50 = 0.5727 pM; SI-55H11 EC50 = 0.09387 pM) compared to the parental penta-GNC antibody lacking one of the tumor target domains. Therefore, the additional antitumor antigen-binding domain can enhance the biological function of GNC antibodies even against cells expressing low levels of TAA (in this case, HER3).

[0125] The above description and examples provide a complete description of the structure and use of exemplary embodiments. While specific embodiments have been described above with some degree of specificity or by reference to one or more individual embodiments, those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the scope of the invention. For example, the above examples may include binding domains at specific locations, but these are provided for comparison purposes only and not as limitations. It is specifically considered herein that the configuration of binding domains and their locations on GNC proteins can be in any combination. Thus, the exemplary embodiments of the invention are not intended to be limited to any specific embodiments disclosed. Rather, they include all modifications and substitutions within the scope of the disclosure. Furthermore, where necessary, aspects of any of the above examples can be combined with any of the other examples described to form further examples having equivalent or different characteristics and addressing the same or different problems. Similarly, it will be understood that the above advantages and benefits may relate to one embodiment or to several embodiments.

[0126] [Table 1]

[0127] a See 284A10, applicant's application number PCT / US2018 / 039143. b 284A10 H1 (SEQ ID NOs: 93-100), a humanized anti-CD3 variable domain sequence (SEQ ID NOs: 155-108) encoding an scFv domain or Fab region in a "non-staple" or "staple" form. c 283E3 H1 (SEQ ID NOs: 101-104), a humanized anti-CD3 variable domain sequence encoding the Fab region.

[0128] dSI-huBU12 VH (SEQ ID NOs. 121, 122) and SI-huBU12 H1 VL (SEQ ID NOs. 87, 88), humanized anti-CD19 variable domain sequences with R19S mutations.

[0129] e SI-huBU12 H1 VH (sequence codes 85, 86) and SI-huBU12 H1 VL (sequence codes 87, 88), humanized anti-CD19 variable domain sequences.

[0130] f SI-huBU12 VH (SEQ ID NOs. 121, 122) and SI-huBU12 VL (SEQ ID NOs. 131, 132), humanized anti-CD19 variable domain sequences with R19S mutations.

[0131] [Table 2]

[0132] HMW% was measured by preparative SEC. Melting temperature was measured by dynamic light scattering.

[0133] [Table 3]

[0134] [Table 4] [Table 5]

[0135] [Table 6]

[0136] [Table 7]

[0137] [Table 8] *Mouse sequence derived from cetuximab Sequence List JPEG0007918236000009.jpg134101JPEG0007918236000010.jpg35101 >Sequence ID 1: SI-1P1 heavy chain amino acid sequence >Sequence ID 2: SI-1P1 heavy chain nucleotide sequence >Sequence ID 3: SI-1P1 light chain moiety amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 4: SI-1P1 light chain moiety nucleotide sequence >Sequence ID 5: SI-1P2 heavy chain amino acid sequence >Sequence ID 6: SI-1P2 heavy chain nucleotide sequence >Sequence ID 7: SI-1P2 light chain moiety amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGCGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 8: SI-1P2 light chain moiety nucleotide sequence >Sequence ID 9: SI-38P12 heavy chain amino acid sequence >Sequence ID 10: SI-38P12 heavy chain nucleotide sequence >Sequence ID 11: SI-38P12 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 12: SI-38P12 light chain moiety nucleotide sequence >Sequence ID 13: SI-38P13 heavy chain amino acid sequence >Sequence ID 14: SI-38P13 heavy chain nucleotide sequence >Sequence ID 15: SI-38P13 light chain moiety amino acid sequence DPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVL >Sequence ID 16: SI-38P13 light chain moiety nucleotide sequence >Sequence ID 17: SI-49P1 heavy chain amino acid sequence >Sequence ID 18: SI-49P1 heavy chain nucleotide sequence >Sequence ID 19: SI-49P1 light chain moiety amino acid sequence DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV >Sequence ID 20: SI-49P1 light chain moiety nucleotide sequence >Sequence ID 21: SI-49P3 heavy chain amino acid sequence >Sequence ID 22: SI-49P3 heavy chain nucleotide sequence >Sequence ID 23: SI-49P3 light chain moiety amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV >Sequence ID 24: SI-49P3 light chain moiety nucleotide sequence >Sequence ID 25: SI-55P3 heavy chain amino acid sequence >Sequence ID 26: SI-55P3 heavy chain nucleotide sequence >Sequence ID 27: SI-55P3 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 28: SI-55P3 light chain moiety nucleotide sequence >Sequence ID 29: SI-55P4 heavy chain amino acid sequence >Sequence ID 30: SI-55P4 heavy chain nucleotide sequence >Sequence ID 31: SI-55P4 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 32: SI-55P4 light chain moiety nucleotide sequence >Sequence ID 33: SI-55P9 heavy chain amino acid sequence >Sequence ID 34: SI-55P9 heavy chain nucleotide sequence >Sequence ID 35: SI-55P9 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 36: SI-55P9 light chain moiety nucleotide sequence >Sequence ID 37: SI-55P10 heavy chain amino acid sequence >Sequence ID 38: SI-55P10 heavy chain nucleotide sequence >Sequence ID 39: SI-55P10 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 40: SI-55P10 light chain moiety nucleotide sequence >Sequence ID 41: SI-77P1 heavy chain amino acid sequence >Sequence ID 42: SI-77P1 heavy chain nucleotide sequence >Sequence ID 43: SI-77P1 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 44: SI-77P1 light chain moiety nucleotide sequence >Sequence ID 45: SI-79P2 heavy chain amino acid sequence >Sequence ID 46: SI-79P2 heavy chain nucleotide sequence >Sequence ID 47: SI-79P2 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 48: SI-79P2 light chain moiety nucleotide sequence >Sequence ID 49: SI-79P3 heavy chain amino acid sequence >Sequence ID 50: SI-79P3 heavy chain nucleotide sequence >Sequence ID 51: SI-79P3 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 52: SI-79P3 light chain moiety nucleotide sequence >Sequence ID 53: SI-55H11 heavy chain amino acid sequence EIVLTQSPSTLSVSPGERATFSC RASQSIGTNIH WYQQKPGKPPRLLIK YASESIS GIPDRFSGSGSGTEFTLTISSVQSEDFAVYYC QQNNNWPTT FGPGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLT NYGVH WIRQAPGKGLEWLG VIWSGGNTDYNTPFTS RFTITKDNSKNQVYFKLRSVRADDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVREAPGKCLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK >Sequence ID 54: SI-55H11 heavy chain nucleotide sequence >Sequence ID 55: SI-55H11 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITC SASSSVSYM HWYQQKPGQAPKLWIY DTSKLAS GVPSRFSGSGSGNDHTLTISSMEPEDFATYYC FQGSVYPFT FGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLS TSGMG VG WIRQPPGKGLEWLA HIWWDDDKRYNPALKS RLTISKDTSKNQVYLQMNSLDAEDTAVYYCAR MELWSYYFDY WGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGCGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISC TGTSSDVGGYNFVS WYQQHPGKAPKLMIY DVSDRPS GVSDRFSGSKSGNTASLIISGLQADDEADYYC SSYGSSSTHVI FGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINRDGSASYYVDSVKG RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR DRGVGYFDL WGRGTLVTVSS >Sequence ID 56: SI-55H11 light chain moiety nucleotide sequence >Sequence ID 57: SI-55H12 heavy chain amino acid sequence >Sequence ID 58: SI-55H12 heavy chain nucleotide sequence >Sequence ID 59: SI-55H12 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 60: SI-55H12 light chain moiety nucleotide sequence >Sequence ID 61: SI-77H4 heavy chain amino acid sequence >Sequence ID 62: SI-77H4 heavy chain nucleotide sequence >Sequence ID 63: SI-77H4 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGCGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 64: SI-77H4 light chain moiety nucleotide sequence >Sequence ID 65: SI-77H5 heavy chain amino acid sequence >Sequence ID 66: SI-77H5 heavy chain nucleotide sequence >Sequence ID 67: SI-77H5 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 68: SI-77H5 light chain moiety nucleotide sequence >Sequence ID 69: αEGFR H1 VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCKVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 70: αEGFR H1 VH nucleotide sequence GAAGTTCAGCTGGTGGAATCCGGCGGAGGATTGGTTCAACCTGGCGGCTCTCTGAGACTGTCCTGTAAGGTGTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTCCGACAGGCACCTGGAAAAGGACTGGAATGGGTCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCTCGGGACACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTAGAGCCCTGACCTACTATGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 71: αEGFR H1 VL amino acid sequence EIVMTQSPSTLSASVGDRVIITCRASQSIGTNIHWYQQKPGKAPKLLIYYASESISGIPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQNNNWPTTFGQGTKLTVL >Sequence ID 72: αEGFR H1 VL nucleotide sequence GAGATCGTGATGACCCAGTCTCCTTCCACACTGTCCGCCTCTGTGGGCGACAGAGTGATCATCACCTGTAGAGCCAGCCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGATCTGGCGCTGAGTTTACCCTGACAATCTCCAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAACTGACAGTTCTT >Sequence ID 73: αEGFR H4 VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 74: αEGFR H4 VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Sequence ID 75: αEGFR H4 VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLELK >Sequence ID 76: αEGFR H4 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAATTGAAA >Sequence ID 77: αEGFR H7 VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 78: αEGFR H7 VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Sequence ID 79: αEGFR H7 VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVL >Sequence ID 80: αEGFR H7 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACAGTTCTT >Sequence ID 81: αEGFR H7 VH staple amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKCLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 82: αEGFR H7 VH staple nucleotide sequence CAAGTACAGTTGCAGCAATCCGGTCCCGGTCTCGTCAAACCGAGTGAGACGCTTAGTATAACGTGTACTGTTTCAGGCTTTAGCCTTACGAACTATGGAGTTCACTGGATTCGGCAGGCACCCGGCAAATGTTTGGAATGGCTGGGTGTTATTTGGTCAGGTGGAAATACAGACTATAACACCCCCTTTACAAGTCGGTTCACAATTACGAAAGATAATTCCAAAAATCAAGTTTATTTCAAGTTGAGATCCGTCCGCGCGGACGACACTGCGATCTACTATTGTGCGAGGGCACTGACCTACTACGATTACGAATTTGCGTATTGGGGGCAAGGGACTCTTGTAACAGTCTCCAGT >Sequence ID 83: αEGFR H7 VL staple amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGCGTKLTVL >Sequence ID 84: αEGFR H7 VL staple nucleotide sequence GAAATCGTCCTTACACAATCTCCTAGCACACTGAGTGTGAGCCCCGGCGAACGCGCGACTTTCTCTTGCAGGGCAAGTCAATCCATAGGGACTAATATACATTGGTATCAACAAAAGCCAGGTAAACCACCCAGGCTTTTGATTAAGTATGCAAGTGAGTCTATTTCCGGTATCCCTGACCGCTTCTCTGGATCAGGCAGTGGCACAGAGTTCACACTCACCATATCTAGTGTGCAATCAGAGGACTTCGCCGTGTATTACTGCCAACAGAATAATAACTGGCCGACTACCTTCGGATGCGGTACAAAGCTGACCGTTTTA >Sequence ID 85: αCD19 SI-huBU12 H1 VH amino acid sequence QVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSS >Sequence ID 86: αCD19 SI-huBU12 H1 VH nucleotide sequence CAGGTCACATTGAAGGAATCTGGCCCCGGCCTTGTTCAGCCAGGACAGACCCTTAGCCTCACCTGTGCCTTCAGTGGTTTTTCTCTTAGCACTAGCGGTATGGGGGTCGGCTGGATTCGGCAGCCTCCCGGCAAAGGTCTTGAGTGGTTGGCTCACATTTGGTGGGACGACGACAAACGGTATAATCCTGCCTTGAAAAGTCGGCTGACCATTAGTAAGGATACCTCAAAAAATCAAGTGTACTTGCAAATGAATAGCCTTGACGCCGAGGATACGGCTGTATATTATTGCGCTCGGATGGAACTCTGGTCTTACTACTTTGATTATTGGGGGCAGGGGACTCTCGTCACGGTCTCGAGT >Sequence ID 87: αCD19 SI-huBU12 H1 VL amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVL >Sequence ID 88: αCD19 SI-huBU12 H1 VL nucleotide sequence GAAAATGTATTGACACAGAGCCCCGCCTCCCTCAGTGCCTCACCTGGGGAAAGGGTAACTATCACTTGCTCTGCATCAAGCAGCGTCTCATACATGCATTGGTATCAACAAAAGCCTGGACAGGCCCCCAAGCTCTGGATATACGATACGAGCAAGCTGGCTTCCGGCGTACCTAGCCGCTTCAGTGGTTCCGGCTCAGGCAACGATCACACCCTTACGATTTCCAGTATGGAACCCGAAGATTTTGCAACTTATTATTGTTTCCAGGGGAGCGTGTACCCATTCACTTTCGGGCAGGGGACAAAAGTGACCGTCCTA >Sequence ID 89: αCD3 284A10 staple VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTISTNAMSWVRQAPGKCLEWIGVITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVST >Sequence ID 90: αCD3 284A10 staple VH nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGTGGTTCTTCTGCTATTACTAGTAACAACATTTGGGGCCAGGGAACCCTGGTCACCGTGTCGACA >Sequence ID 91: αCD3 284A10 staple VL amino acid sequence DVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGCGTKVEIK >Sequence ID 92: αCD3 284A10 staple VL nucleotide sequence GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCTGTGGGACCAAGGTGGAGATCAAA >Sequence ID 93: αCD3 284A10 H1 VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTISTNAMSWVRQAPGKGLEWVGVITGRDITYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSS >Sequence ID 94: αCD3 284A10 H1 VH nucleotide sequence GAAGTCCAATTGGTAGAAAGTGGCGGTGGTCTGGTGCAACCTGGTGGATCTCTTCGCCTCTCATGCGCCGCTAGTGGCTTTACTATTTCAACTAATGCGATGAGCTGGGTTCGCCAGGCCCCCGGCAAAGGACTTGAGTGGGTCGGCGTCATCACCGGCAGGGACATTACATACTATGCGAGTTGGGCAAAGGGCAGGTTCACGATTAGCCGCGATACTTCAAAGAATACCGTTTACCTTCAAATGAATAGCTTGAGGGCGGAAGACACAGCTGTGTATTACTGCGCGAGGGATGGAGGTAGTTCCGCCATAACTTCCAACAACATATGGGGACAAGGCACGCTGGTTACTGTCTCGAGT >Sequence ID 95: αCD3 284A10 H1 VL amino acid sequence EIVMTQSPSTLSASVGDRVIITCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGQGTKLTVL >Sequence ID 96: αCD3 284A10 H1 VL nucleotide sequence GAAATCGTTATGACGCAGAGTCCCTCCACGCTCTCCGCTAGTGTCGGGGATCGCGTCATTATCACATGCCAGGCCTCCGAGTCAATCAGCAGCTGGCTTGCATGGTATCAACAGAAGCCGGGAAAAGCTCCTAAATTGCTGATCTATGAAGCGTCAAAATTGGCGTCTGGTGTCCCATCTAGGTTCTCCGGCTCTGGGTCTGGTGCGGAATTTACTTTGACAATCTCCAGTCTTCAACCAGACGATTTCGCTACCTACTACTGCCAAGGGTATTTCTATTTTATAAGCCGGACATATGTAAACTCCTTCGGCCAAGGAACAAAGTTGACTGTTCTT >Sequence ID 97: αCD3 284A10 H1 staple VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGFTISTNAMSWVRQAPGKCLEWVGVITGRDITYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVST >Sequence ID 98: αCD3 284A10 H1 staple VH nucleotide sequence GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCACCGCCAGCGGCTTCACCATCAGCACCAACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGGCGTGATCACCGGCAGGGACATCACCTACTACGCCAGCTGGGCCAAGGGCAGGTTCACCATCAGCAGGGACACCAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGCGGCAGCAGCGCCATCACCAGCAACAACATCTGGGGCCAGGGCACCCTGGTGACCGTGTCGACA >Sequence ID 99: αCD3 284A10 H1 staple VL amino acid sequence EIVMTQSPSTLSASVGDRVIITCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGCGTKLTVL >Sequence ID 100: αCD3 284A10 H1 staple VL nucleotide sequence GAGATCGTGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGATCATCACCTGCCAGGCCAGCGAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGCAAGCTGGCCAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCGCCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGGGCTACTTCTACTTCATCAGCAGGACCTACGTGAACAGCTTCGGCTGCGGCACCAAGCTGACCGTGCTG >Sequence ID 101: αCD3 283E3 H1 VH amino acid sequence QVQLQESGGRLVQPGEPLSLTCKTSGIDLSSNAIGWVRQAPGKGLEWIGVIFGSGNTYYASWAKGRFTISRSTSTVYLKMNSLRSEDTAIYYCARGGYSSDIWGQGTLVTVSS >Sequence ID 102: αCD3 283E3 H1 VH nucleotide sequence CAAGTGCAGTTGCAAGAAAGTGGTGGTAGACTGGTTCAGCCTGGTGAACCCTTGTCACTGACGTGTAAAACAAGCGGCATTGATCTGTCCTCTAACGCCATCGGATGGGTCCGACAGGCCCCAGGAAAAGGTCTGGAGTGGATCGGAGTTATCTTCGGGAGCGGCAATACATACTACGCAAGCTGGGCAAAAGGGCGATTTACGATATCACGGAGCACCTCTACAGTTTATTTGAAAATGAACTCCCTCCGGTCCGAGGATACCGCGATATATTACTGTGCCAGAGGGGGGTACTCCTCTGATATCTGGGGGCAGGGTACACTGGTTACAGTTTCATCC >Sequence ID 103: αCD3 283E3 H1 VL amino acid sequence DPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIK >Sequence ID 104: αCD3 283E3 H1 VL nucleotide sequence GATCCAGTTCTGACACAAAGTCCATCCAGCCTGTCTGCCTCAGTCGGCGACAGAGTGACCATCAGTTGCCAGAGCTCACAGTCTGTGGCTAAGAACAACAACTTGGCGTGGTTCCAACAGAAACCTGGACAGGCTCCGAAATTGCTGATCTATTCTGCTTCCACGCTTGCTGCTGGTGTTCCTTCCCGCTTTTCAGGTAGTGGTAGCGGGACAGACTTCACTTTGACTATAAGCAGCGTGCAGCCTGAAGATTTTGCGACCTACTATTGTTCTGCTAGAGACAGTGGAAATATTCAGTCCTTTGGGGGGGGAACGAAGGTCGAAATAAAG >Sequence ID 105: αPDL1 PL221G5 staple VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKCLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSS >Sequence ID 106: αPDL1 PL221G5 staple VH nucleotide sequence GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTACGACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC >Sequence ID 107: αPDL1 PL221G5 staple VL amino acid sequence DIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWGNVDNVFGCGTKVEIK >Sequence ID 108: αPDL1 PL221G5 staple VL nucleotide sequence GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCTGCGGGACCAAGGTGGAGATCAAA >Sequence ID 109: α41BB 466F6 staple VH amino acid sequence RSLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKCLEYIGTISSGGNVYYASSARGRFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSS >Sequence ID 110: α41BB 466F6 staple VH nucleotide sequence CGGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCACCATCAGTAGCTACCACATGCAGTGGGTCCGGCAGGCACCTGGGAAGTGCCTGGAGTACATCGGAACCATTAGTAGTGGTGGTAATGTATACTACGCAAGCTCCGCTAGAGGCAGATTCACCATCTCCAGACCCTCGTCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTCTGGTTATAGTGATCCTATGTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA >Sequence ID 111: α41BB 466F6 staple VL amino acid sequence DVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGSGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGCGTKVEIK >Sequence ID 112: α41BB 466F6 staple VL nucleotide sequence GACGTTGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACCTGTCAGGCCAGTCAGAACATTAGGACTTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAGCCAATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGACTTGGAACCTGGCGATGCTGCAACTTACTATTGTCAGTCTACCTATCTTGGTACTGATTATGTTGGCGGTGCTTTCGGCTGTGGGACCAAGGTGGAGATCAAATGA >Sequence ID 113: 4-1BB ligand trimer amino acid sequence REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGSTGSGSKPGSGEGSTKGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL >Sequence ID 114: 4-1BB ligand trimer nucleotide sequence >Sequence ID 115: NKG2D dimer amino acid sequence FLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV >Sequence ID 116: NKG2D dimer nucleotide sequence TTTCTTAATTCCCTTTTCAACCAAGAGGTTCAGATCCCCTTGACTGAAAGCTATTGCGGCCCTTGTCCGAAAAACTGGATATGTTACAAGAATAATTGTTACCAATTCTTCGACGAAAGCAAGAACTGGTATGAGAGTCAGGCGTCTTGTATGAGTCAGAATGCCAGCCTGCTTAAGGTTTATTCAAAAGAAGACCAGGATCTGCTTAAGTTGGTAAAGAGCTACCACTGGATGGGGCTGGTACATATCCCAACGAATGGGTCATGGCAGTGGGAGGACGGTTCTATTCTGAGTCCAAATCTCCTGACGATCATCGAAATGCAGAAAGGGGACTGTGCCCTGTATGCATCATCCTTCAAGGGGTACATCGAGAACTGCAGTACCCCAAATACCTACATTTGTATGCAAAGAACGGTTGGAGGCGGTGGCTCAGGCGGAGGCGGCTCAGGAGGTGGCGGTTCAGGAGGCGGCGGATCTTTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTGTAG >Sequence ID 117: SI-49P10 heavy chain amino acid sequence >Sequence ID 118: SI-49P10 heavy chain nucleotide sequence >Sequence ID 119: SI-49P10 light chain moiety amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGSTGSGSKPGSGEGSTKGQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 120: SI-49P10 light chain moiety nucleotide sequence >Sequence ID 121: αCD19 SI-huBU12 VH amino acid sequence QVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSS >Sequence ID 122: αCD19 SI-huBU12 VH nucleotide sequence CAGGTCACATTGAAGGAATCTGGCCCCGGCCTTGTTCAGCCAGGACAGACCCTTAGGCTCACCTGTGCCTTCAGTGGTTTTTCTCTTAGCACTAGCGGTATGGGGGTCGGCTGGATTCGGCAGCCTCCCGGCAAAGGTCTTGAGTGGTTGGCTCACATTTGGTGGGACGACGACAAACGGTATAATCCTGCCTTGAAAAGTCGGCTGACCATTAGTAAGGATACCTCAAAAAATCAAGTGTACTTGCAAATGAATAGCCTTGACGCCGAGGATACGGCTGTATATTATTGCGCGCGGATGGAACTCTGGTCTTACTACTTTGATTATTGGGGGCAGGGGACTCTCGTCACGGTCTCGAGC >Sequence ID 123: SI-49P6 heavy chain amino acid sequence >Sequence ID 124: SI-49P6 heavy chain nucleotide sequence >Sequence ID 125: SI-49P6 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKLEIKGSTGSGSKPGSGEGSTKGQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 126: SI-49P6 light chain moiety nucleotide sequence >Sequence ID 127: SI-49P7 heavy chain amino acid sequence >Sequence ID 128: SI-49P7 heavy chain nucleotide sequence >Sequence ID 129: SI-49P7 light chain moiety amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKLEIKGSTGSGSKPGSGEGSTKGQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 130: SI-49P7 light chain moiety nucleotide sequence >Sequence ID 131: αCD19 SI-huBU12 VL amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKLEIK >Sequence ID 132: αCD19 SI-huBU12 VL nucleotide sequence GAAAATGTATTGACACAGAGCCCCGCCTCCCTCAGTGCCTCACCTGGGGAAAGGGTAACTATCACTTGCTCTGCATCAAGCAGCGTCTCATACATGCATTGGTATCAACAAAAGCCTGGACAGGCCCCCAAGCTCTGGATATACGATACGAGCAAGCTGGCTTCCGGCGTACCTAGCCGCTTCAGTGGTTCCGGCTCAGGCAACGATCACACCCTTACGATTTCCAGTATGGAACCCGAAGATTTTGCAACTTATTATTGTTTCCAGGGGAGCGTGTACCCATTCACTTTCGGGCAGGGGACAAAATTGGAGATAAAG

Claims

1. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The aforementioned D2 includes a heavy chain portion and a light chain portion, and a fifth binding domain (D5) is covalently bonded to the C-terminus of the light chain portion. The multispecific antibody-like protein has D1 binding specificity to CD3, D2 binding specificity to EGFR, D3 binding specificity to PD-L1, D4 binding specificity to 4-1BB, and D5 binding specificity to HER3. The multispecific antibody-like protein comprises the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 3, or the heavy chain amino acid sequence shown in SEQ ID NO: 5 and the light chain amino acid sequence shown in SEQ ID NO:

7. Multiple specificity antibody-like proteins.

2. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The aforementioned D2 includes a heavy chain portion and a light chain portion, and a sixth binding domain (D6) is covalently bonded to the N-terminus of the light chain portion. The multispecific antibody-like protein has D1 binding specificity to CD20, D2 binding specificity to CD3, D3 binding specificity to PD-L1, D4 binding specificity to 4-1BB, and D6 binding specificity to CD19. The multispecific antibody-like protein includes the heavy chain amino acid sequence shown in SEQ ID NO: 9 and the light chain amino acid sequence shown in SEQ ID NO:

11. Multiple specificity antibody-like proteins.

3. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The aforementioned D2 includes a heavy chain portion and a light chain portion, and a fifth binding domain (D5) is covalently bonded to the C-terminus of the light chain portion. The multispecific antibody-like protein has D1 binding specificity to CD20, D2 binding specificity to CD3, D3 binding specificity to PD-L1, D4 binding specificity to 4-1BB, and D5 binding specificity to CD19. The multispecific antibody-like protein includes the heavy chain amino acid sequence shown in SEQ ID NO: 13 and the light chain amino acid sequence shown in SEQ ID NO:

15. Multiple specificity antibody-like proteins.

4. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The aforementioned D2 includes a heavy chain portion and a light chain portion, and a sixth binding domain (D6) is covalently bonded to the N-terminus of the light chain portion. The multispecific antibody-like protein has D1 binding specificity to EGFR, D2 binding specificity to CD3, D3 binding specificity to PD-L1, D4 binding specificity to 4-1BB, and D6 binding specificity to CD19. The multispecific antibody-like protein includes the heavy chain amino acid sequence shown in SEQ ID NO: 25 and the light chain amino acid sequence shown in SEQ ID NO: 27, the heavy chain amino acid sequence shown in SEQ ID NO: 29 and the light chain amino acid sequence shown in SEQ ID NO: 31, the heavy chain amino acid sequence shown in SEQ ID NO: 33 and the light chain amino acid sequence shown in SEQ ID NO: 35, the heavy chain amino acid sequence shown in SEQ ID NO: 37 and the light chain amino acid sequence shown in SEQ ID NO: 39, the heavy chain amino acid sequence shown in SEQ ID NO: 45 and the light chain amino acid sequence shown in SEQ ID NO: 47, or the heavy chain amino acid sequence shown in SEQ ID NO: 49 and the light chain amino acid sequence shown in SEQ ID NO:

51. Multiple specificity antibody-like proteins.

5. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The aforementioned D2 includes a heavy chain portion and a light chain portion, and a sixth binding domain (D6) is covalently bonded to the N-terminus of the light chain portion. The aforementioned multispecific antibody-like protein has D1 which has binding specificity to CD3 and D2 which has EGFR It has binding specificity to , D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, and D6 has binding specificity to CD19. The multispecific antibody-like protein includes the heavy chain amino acid sequence shown in SEQ ID NO: 41 and the light chain amino acid sequence shown in SEQ ID NO:

43. Multiple specificity antibody-like proteins.

6. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The D2 comprises a heavy chain portion and a light chain portion, with a fifth binding domain (D5) covalently bonded to the C-terminus of the light chain portion and a sixth binding domain (D6) covalently bonded to the N-terminus of the light chain portion. The multispecific antibody-like protein has the following characteristics: D1 has binding specificity to EGFR, D2 has binding specificity to CD3, D3 has binding specificity to PD-L1, D4 has binding specificity to 4-1BB, D5 has binding specificity to HER3, and D6 has binding specificity to CD19. The multispecific antibody-like protein comprises the heavy chain amino acid sequence shown in SEQ ID NO: 53 and the light chain amino acid sequence shown in SEQ ID NO: 55, or the heavy chain amino acid sequence shown in SEQ ID NO: 57 and the light chain amino acid sequence shown in SEQ ID NO:

59. Multiple specificity antibody-like proteins.

7. It is a multispecific antibody-like protein, From the N-terminus to the C-terminus, The first binding domain (D1) located at the N-terminus, The Fab region is the second binding domain (D2), Fc region and, The third binding domain (D3), The fourth binding domain (D4) located at the C-terminus, Includes, The D2 comprises a heavy chain portion and a light chain portion, with a fifth binding domain (D5) covalently bonded to the C-terminus of the light chain portion and a sixth binding domain (D6) covalently bonded to the N-terminus of the light chain portion. The multispecific antibody-like protein has D1 binding specificity to CD3, D2 binding specificity to EGFR, D3 binding specificity to PD-L1, D4 binding specificity to 4-1BB, D5 binding specificity to HER3, and D6 binding specificity to CD19. The multispecific antibody-like protein comprises the heavy chain amino acid sequence shown in SEQ ID NO: 61 and the light chain amino acid sequence shown in SEQ ID NO: 63, or the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO:

67. Multiple specificity antibody-like proteins.

8. A guidance and navigation control protein comprising a dimer of a multispecific antibody-like protein according to any one of claims 1 to 7.

9. An isolated nucleic acid encoding the amino acid sequence of a multispecific antibody-like protein according to any one of claims 1 to 7.

10. An expression vector comprising the isolated nucleic acid described in claim 9.

11. A host cell comprising the isolated nucleic acid described in claim 9, The host cell is a host cell that is either a prokaryotic cell or a eukaryotic cell.

12. The steps include culturing a host cell containing isolated nucleic acid so that a DNA sequence encoding a multispecific antibody-like protein according to any one of claims 1 to 7 is expressed, The step of purifying the multispecific antibody-like protein, A method for producing multispecific antibody-like proteins, including

13. Use of a multispecific antibody-like protein according to any one of claims 1 to 7 in the manufacture of a drug for treating or preventing cancer, autoimmune disease, or infectious disease.

14. A drug for treating or preventing cancer, autoimmune disease, or infectious disease, comprising a multispecific antibody-like protein according to any one of claims 1 to 7.

15. The cytotoxic agent or imaging agent is conjugated via a linker to the multispecific antibody-like protein described in any one of claims 1 to 7, The linker is an immunocomplex comprising an ester bond, an ether bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphate ester bond, a peptide bond, a hydrophobic poly(ethylene glycol) linker, or a combination thereof.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a multispecific antibody-like protein according to any one of claims 1 to 7 and / or an immune complex according to claim 15.

Images