Guidance and navigation control proteins, and methods of making and using thereof

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 TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 931,307, filed November 6, 2019, U.S. Provisional Application No. 62 / 984,731, filed March 3, 2020, and U.S. Provisional Application No. 62 / 991,042, filed March 17, 2020 (35 U.S.C. 119(e)), the entire disclosures of which are incorporated herein by reference.

[0002] This application relates to the technical field of multispecific antibodies for use in immunotherapy, and more particularly to methods for making and using guidance and navigation control (GNC) antibodies with multibinding activity for surface molecules of immune cells and tumor cells. [Background technology]

[0003] Cancer progresses by acquiring mutations that allow cancer cells to transform, grow, and metastasize while evading immune surveillance and response. Antibody therapies for cancer treatment employ several different mechanisms. For example, monoclonal antibodies targeting growth factor receptors (e.g., EGFR, HER2) overexpressed on tumor cells can be used to block tumor cell proliferation. Blocking inhibitory T cell checkpoint signals (anti-PDL1, anti-PD1, anti-CTLA4) with antibodies is a way to prevent tumor cells from growing by weakening the immune response that controls tumor cell growth. 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 tumor control. However, cancer resistance to antibody therapy often occurs through escape mechanisms such as ectodomain shedding, receptor downregulation, and receptor mutations (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 can occur through HER2 ectodomain shedding or blockage 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, radiation therapy, and antibody therapy, has become a mainstream treatment strategy. Herein, multispecific antibodies combine different antibody therapies and mechanisms into a single agent (Boumahdi and de Sauvage. Nat Rev Drug Discov. 2020). Summary of the Invention

[0005] The following summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0006] In one aspect, the present invention provides a guidance and navigation control (GNC) protein capable of simultaneously binding to effector cells and target cells. The GNC protein may be a monomer or a dimer of monomers. The GNC protein may be an antibody or 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, in order from N-terminus to C-terminus, a first binding domain (D1) located at the N-terminus, a second binding domain (D2) comprising 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 comprises a fifth binding domain (D5) covalently linked to the C-terminus and / or a sixth binding domain (D6) covalently linked to the N-terminus. D1, D2, D3, D4, D5, and D6 each have binding specificity for a tumor antigen, an immune signaling antigen, or a combination thereof.

[0008] The tumor antigen may be a tissue antigen, a neoantigen, a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), or a combination thereof.

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

[0010] D2 may comprise a dimer.

[0011] In one embodiment, D2 may comprise a Fab region. In one embodiment, the Fab region comprises a V L and V H In one embodiment, D2 may have a disulfide bond between V L and V H may include:

[0012] In one embodiment, D2 may comprise a receptor. In one embodiment, the receptor may be NKG2D. In one embodiment, D2 may comprise C H1 and C L 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 to SEQ ID NOs: 155 and 116.

[0013] D2 may be linked to the 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 a null Fc. In one embodiment, the LALAKA mutations for a null Fc may comprise L234A / L235A / K322A (Eu numbering) mutations. 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, the present invention provides a multispecific antibody-like protein having an N-terminus and a C-terminus, which comprises, in order from N-terminus to C-terminus, a first binding domain (D1) located at the N-terminus, a second binding domain (D2) located at the C-terminus, and a third binding domain (D3) located at the C-terminus. L and C H1 The second binding domain (D2) contains a dimer bound to C via a hinge. H1and a fourth binding domain (D4) located at the C-terminus. The light chain portion may have a fifth binding domain (D5) covalently attached to the C-terminus and / or a sixth binding domain (D6) covalently attached to the N-terminus. D1, D2, D3, D4, D5, and D6 may each have binding specificity for a tumor antigen, an immune signaling antigen, or a combination thereof.

[0016] The dimers in D2 are C L and C H V bound to L and V H 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 pentaspecific or hexaspecific.

[0018] In one embodiment, the light chain portion of D2 may have a fifth binding domain (D5) covalently attached to the C-terminus, and the multispecific antibody-like protein is pentaspecific. In one embodiment, the light chain portion may have a sixth binding domain (D6) covalently attached to the N-terminus, and the multispecific antibody-like protein is pentaspecific. In one embodiment, the light chain portion may simultaneously have both a fifth binding domain (D5) covalently attached to the C-terminus and a sixth binding domain (D6) covalently attached to the N-terminus, making the multispecific antibody-like protein hexaspecific.

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

[0020] The scFv domain consists of V from the N-terminus to the C-terminus. L V H or V H V LIn one embodiment, the scFv domain may have the R19S (Kabat) mutation. In one embodiment, the scFv domain may have the V L and V H In one embodiment, the disulfide bond is between the V and V of the scFv domain. L 100 and V H 44 (Kabat). In one embodiment, the scFv domain may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 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 independently be a receptor or a 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 comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 113-116.

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

[0024] In one embodiment, D1, D2, D3, D4, D5, and D6 each independently have binding specificity for an antigen selected from a receptor on a T cell, an immune checkpoint receptor, a costimulatory receptor, a receptor on a lymphocyte or myeloid cell, a tumor-associated antigen (TAA), a tissue antigen, a neoantigen, a tumor-specific antigen (TSA), a glycoprotein, and combinations thereof.

[0025] In one embodiment, the binding domain for a receptor on a T cell may be adjacent to a binding domain for a tumor-associated antigen (TAA). In one embodiment, the binding domain for a receptor on a T cell may be adjacent to a binding domain for a receptor on a 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 each independently have binding specificity for an antigen 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. The Fc region comprises a human IgG Fc region.

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

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

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

[0031] In one embodiment, the multispecific antibody-like protein is pentaspecific, where D1 has binding specificity for CD20, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 9-12.

[0032] In one embodiment, the multispecific antibody-like protein is pentaspecific, where D1 has binding specificity for CD20, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D5 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 13-16.

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

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

[0035] In one embodiment, the multispecific antibody-like protein is pentaspecific, where D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 25-28.

[0036] In one embodiment, the multispecific antibody-like protein is pentaspecific, wherein D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 comprises a 4-1BB ligand trimer, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 29-32.

[0037] In one embodiment, the multispecific antibody-like protein is pentaspecific, where D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 33-36.

[0038] In one embodiment, the multispecific antibody-like protein is pentaspecific, wherein D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 comprises a 4-1BB ligand trimer, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 37-40.

[0039] In one embodiment, the multispecific antibody-like protein is pentaspecific, and D1 has binding specificity for CD3, D2 has binding specificity for EGFR, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 41-44.

[0040] In one embodiment, the multispecific antibody-like protein is pentaspecific, where D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 45-48.

[0041] In one embodiment, the multispecific antibody-like protein is pentaspecific, wherein D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 comprises a 4-1BB ligand trimer, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 49-52.

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

[0043] In one embodiment, the multispecific antibody-like protein is pentaspecific, and D1 has binding specificity for CD3, D2 comprises NKG2D, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 123-126.

[0044] In one embodiment, the multispecific antibody-like protein is pentaspecific, and D1 has binding specificity for CD3, D2 comprises NKG2D, D3 has binding specificity for PD-L1, D4 comprises a 4-1BB ligand trimer, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 127-130.

[0045] In one embodiment, the multispecific antibody-like protein is hexaspecific, where D1 has binding specificity for EGFR, D2 has binding specificity for CD3, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, D5 has binding specificity for HER3, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 53-60.

[0046] In one embodiment, the multispecific antibody-like protein is hexaspecific, and D1 has binding specificity for CD3, D2 has binding specificity for EGFR, D3 has binding specificity for PD-L1, D4 has binding specificity for 4-1BB, D5 has binding specificity for HER3, and D6 has binding specificity for CD 19. In one embodiment, the multispecific antibody-like protein comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to 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 a 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 aspect, the present invention provides an isolated nucleic acid sequence encoding the amino acid sequence of a multispecific antibody-like protein, fragment or derivative thereof described herein.

[0050] In one aspect, the present invention provides an expression vector comprising the isolated nucleic acid sequence described above.

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

[0052] In one aspect, the present invention provides a method for producing the GNC protein. In one embodiment, the method for producing a multispecific antibody or monomer comprises 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 aspect, the present invention provides an immunoconjugate comprising a cytotoxic agent or imaging agent conjugated to a GNC protein, such as a multispecific antibody-like protein or a multispecific antibody, via a linker. The linker may comprise a covalent bond, such as 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.

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

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

[0056] In one embodiment, the pharmaceutical composition may further comprise a therapeutic agent selected from a radioisotope, a radionuclide, a toxin, a chemotherapeutic agent, or a combination thereof.

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

[0058] In one aspect, the present invention provides a method for treating a human subject having cancer, an autoimmune disease, or an infectious disease, comprising administering to the subject an effective amount of a GNC protein, such as a purified multispecific antibody, multispecific antibody-like protein, or fragment thereof.

[0059] In one embodiment, the method may further comprise co-administering an effective amount of a therapeutic agent, including an antibody, a chemotherapeutic agent, an enzyme, an anti-estrogen, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a checkpoint inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, an inhibitor of PD1, PD-L1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, or a combination thereof.

[0060] In one aspect, the present invention provides a solution comprising an effective concentration of a GNC protein, such as a multispecific antibody, a multispecific antibody-like protein, or a fragment thereof. In one embodiment, the solution can be plasma from a human subject. [Brief explanation of the drawings]

[0061] The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, which illustrate only some embodiments prepared in accordance with the present disclosure and therefore should not be considered limiting of its scope, the present disclosure may be explained with additional specificity and detail through the use of the accompanying drawings.

[0062] [Figure 1] The schematic structure of the antigen-binding domain is shown in Figure 1A. Figure 1A shows the penta-GNC antibody, and Figure 1B shows the hexa-GNC antibody. In Figure 1, the variable region of the Fab (which can be replaced by a receptor or ligand) is shown in black (D2), the constant region and Fc region of the Fab are shown in white, and the additional scFv antigen-binding domain is shown in a shaded box (each of which can be replaced by a receptor-ligand binding). The heavy chain monomer has D1 attached to its N-terminus, and D3 and D4 attached to its C-terminus via D4. The light chain monomer has D5 and / or D6 attached 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) exerted maximal T cell activation and showed similar efficacy to the tetra-GNC antibody targeting HER3 (tetra) or a non-tumor antigen (tetra, FITC) and a bispecific antibody targeting only the tumor antigen (BI). [Figure 3]We demonstrate the high potency of SI-1P1 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 that lacks binding to HER3, a tetra-GNC control antibody that lacks binding to both tumor antigens, and a bispecific antibody that targets only tumor antigens). [Figure 4] Figure 4 shows the effect of using the NKG2D receptor as a binding domain for the GNC antibody. 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) that express tumor antigens other than EGFR and HER3 (MICA and mesothelin), as well as two control antibodies (a tetra-GNC antibody lacking NKG2D and a tri-GNC antibody lacking binding specificity for both PD-L1 and 4-1BB). [Figure 5] In reporter bioassays using Jurkat cells, the 4-1BBL-trimer-Fc fusion protein mediates stronger activation of 4-1BB signaling than other molecules, including monomeric 4-1BB ligand, monomeric Fc, or anti-4-1BB scFv. [Figure 6] Octet binding analysis of the PentaGNC antibody compared to the humanized anti-huEGFR domain shows that variants of the humanized EGFR binding domain (H1, H4, or H7) maintain tight binding to human EGFR with little position effect, even as scFv domains in SI-55P3, SI-79P2, SI55P9, and SI-79P3 or Fab in SI-77P1. [Figure 7] The potency and EC50 values ​​of the PentaGNC antibody in the 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 figure shows the efficacy of the penta-GNC protein containing NKG2D at position D2 in a TDCC assay using human breast cancer cells (MDA-MB-231; MICA expression) as target cells. The obtained EC50 values ​​are 0.7366 pM for SI-49P6 and 0.1094 pM for SI-49P7. [Figure 9] We demonstrate that the tetra-GNC antibody SI-35E20 induces RTCC in NucRed-transduced lung cancer cells A549 and suppresses lung adenocarcinoma cell proliferation in the presence of PBMCs. [Figure 10] Tetra-GNC antibody SI-38E17 is shown to induce RTCC in Nuc-GFP Nalm-6 leukemia cells in the presence or absence of donor PBMCs. [Figure 11] Tetra-GNC antibody SI-39E18 induces RTCC and kills human bladder cancer-derived NucRed+ UMUC3viii cells in the presence of donor PBMCs versus vehicle control. [Figure 12] By administering JVM-3 cells and donor PBMCs (5 x 106 and 2 x 107, respectively) to NCG mice, we demonstrate that 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 × 10 and 5 × 10, respectively) to NCG mice, we demonstrate that the tetra-GNC antibody SI-39E18 effectively suppresses the growth of human bladder cancer cells in a human tumor xenograft model. [Figure 14] In TDCC assays, the hexaGNC antibody (SI-55H11) mediated more complete elimination of human cervical cancer cells (Hela) compared with a similar trispecific antibody, demonstrating the need to simultaneously target immunomodulatory proteins (e.g., PD-L1 immune checkpoint) and activate 4-1BB. [Figure 15]In a TDCC assay using human pancreatic cancer cells (BxPC3) that express low levels of HER3, the hexaGNC antibody (SI-55H11) shows improved potency compared to the parent pentaGNC antibody (SI-55H9) by further binding to HER3. DETAILED DESCRIPTION OF THE INVENTION

[0063] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in various configurations expressly contemplated herein.

[0064] The present invention relates to guidance and navigation control (GNC) proteins, methods for making and 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 referred to as a GNC antibody. In some embodiments, the present invention provides pentaspecific antibody-like proteins and hexaspecific antibody-like proteins. In some embodiments, the present invention provides pentaspecific antibodies and hexaspecific antibodies.

[0065] GNC proteins include proteins that bind multiple functionally independent binding moieties to a single entity that serves both effector cells and target cells (see applicant's application WO / 2019 / 005642, incorporated herein in its entirety). In one embodiment, these multispecific binding molecules target tumor antigens and immune activating receptors, allowing for mechanisms similar to immune effector cell-mediated tumor killing at a fraction of the cost. Rather than genetically modifying individual patient T cells, such multispecific binding molecules can be efficiently manufactured on a large scale and administered in a more conventional, off-the-shelf manner. Among GNC proteins, multispecific antibodies, such as tetrabodies, have been shown to be capable of exerting desirable multifaceted GNC effects, possessing structurally and functionally diverse yet relatively independent binding domains (see applicant's application WO / 2019 / 191120, incorporated herein 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 is composed of one constant domain and one variable domain from the heavy chain portion and the light chain portion. The heavy chain may comprise three additional antigen-specific binding domains attached to the N-terminus and / or C-terminus. The light chain may comprise one or two additional binding domains attached 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 FIG. 1. The GNC antibody may have the ability to attract immune cells (or other effector cells) to tumor cells (or other target cells) through binding of multiple surface molecules on the 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), neoantigens, and tumor-associated antigens (TAAs).

[0068] In one embodiment, the GNC antibody may comprise 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 a T cell comprises a signaling protein, including, but not limited to, CD3, NKG2D, and 4-1BB. The surface molecule on an NK cell comprises a signaling protein, including, but not limited to, NKG2D and 4-1BB. The surface antigen on a tumor cell comprises a tumor antigen, including, but not limited to, EGFR, HER2, HER3, MSLN, CD19, and PD-L1. In one embodiment, the tumor cell comprises a tumor or cancer, including, but 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 the binding specificity and affinity of each binding domain. Each additional binding domain (G x S y ) n It may also include a peptide linker, where 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, a binding domain, e.g., D1, D2, D3, D4, D5, or D6, may be a single chain variable fragment (scFv), receptor, or ligand (Table 1). H -V L (HL) or V L -V H Heavy chain (V) in either (LH) or (LH) orientation H ) and light chain (V L In one embodiment, the scFv domain can be constructed to have a fusion of the variable region of V H 44 and V L A stapled structure may be formed by introducing a disulfide bond between the V 100 (Kabat) and V 200 (Kabat) amino acids. In one embodiment, the V 100 on either light chain portion may be H3 V for containing scFv H The region harbors the R19S mutation (Kabat numbering).

[0071] The binding domain can be configured to bind to at least one epitope of an antigen, including, but 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 can 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 comprise NKG2D.

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

[0074] As used herein, the terms "a," "an," and "the" are defined to mean "one or more," and include the plural referents unless the context is inappropriate.

[0075] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, chimeric, single-chain, multispecific, multipotent, 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 the products of Fab immunoglobulin expression libraries, and epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunoreactive portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. 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 whole antibodies and any antigen-binding fragments derived from whole antibodies. A typical antibody generally refers to a heterotetrameric protein consisting of two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable domain (V H Each light chain portion is composed of a light chain variable domain (V LV H Area and V L The region can be further divided into domains of hypervariable complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs). H or V L ) is usually composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding regions that interact with antigens.

[0076] As used herein, the term "monoclonal" antibody includes "monoclonal monospecific," "chimeric," and "multispecific" antibodies (immunoglobulins), as well as fragments of these antibodies (so long as they exhibit the desired biological activity), in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from other species or belonging to other antibody classes or subclasses (U.S. Pat. No. 4,816,567; Morrison et al., PNAS USA, 1984). Monoclonal antibodies can be prepared by a variety of methods, including mouse hybridomas, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery (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 having binding affinity for an epitope of an antigen. As used herein, the term "bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific" antibody refers to an antibody having two, three, four, five, or six antigen-binding sites. For example, an antibody described herein is pentaspecific if it has five binding sites, and hexaspecific 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 adopt an antibody core structure comprising an Fab region and an Fc region with various binding domains attached to the antibody core. In this case, the GNC protein is also referred to as a GNC antibody. The GNC protein may also adopt 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, an immune cell, or a microbial cell). As used herein, a "bi-GNC, tri-GNC, tetra-GNC, penta-GNC, or hexa-GNC" antibody refers to a GNC antibody having two, three, four, five, or six antigen-binding sites, wherein at least one antigen-binding site has binding affinity for immune cells and at least one antigen-binding site has binding affinity for tumor cells. In one embodiment, the GNC antibodies described herein have five and six binding sites (or binding domains), respectively, and are penta-GNC and hexa-GNC antibodies. In some embodiments, the GNC antibody comprises 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 comprise an engineered Fc region to eliminate effector cell functions, such as ADCC, ADCP, and CDC. Mutations include, but are not limited to, L234A / L235A / G237A / K322A and L234A / L235A / K322A (Eu numbering). In one embodiment, mutations at Fc glycosylation sites, such as N297A (Eu), can 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 into asymmetric molecules. In one embodiment, the GNC antibody further has the advantage of retaining the bivalency of each target antigen. In a further embodiment, the GNC antibody has the advantage of an avidity effect, which results in high affinity for the antigen and a low dissociation rate. This bivalency for each antigen differs from many multispecific platforms, which are monovalent for each target antigen and therefore often lose the beneficial avidity effect that makes antibody binding so strong.

[0080] The term "humanized antibody" refers to an engineered antibody having CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one or more human immunoglobulins. Additionally, framework support residues can be altered to retain binding affinity. 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, "humanized antibodies" can be obtained by genetic engineering methods capable of producing affinity-matured, human-like polyclonal antibodies in large animals (e.g., rabbits) (US Pat. No. 7,129,084).

[0081] The term "antigen" refers to an entity or fragment thereof that is capable of inducing an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0082] The term "epitope", also known as "antigenic determinant", is the part of an antigen that is recognized by the immune system, particularly antibodies, B cells, or T cells, and is the specific part 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 against the immunogenic substance and contributes to a human or animal immune response. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells against the administered immunogenic composition of the invention to alleviate or ameliorate the disorder being treated.

[0084] As used herein, the term "tumor antigen" refers to an antigenic molecule produced by a tumor cell. A tumor antigen can elicit an immune response in a host. In one embodiment, a tumor cell expresses a tumor antigen, including, but not limited to, tumor-specific antigens (TSAs), neoantigens, and tumor-associated antigens (TAAs).

[0085] As used herein, the terms "binds specifically," "specific binding," or "specific" for a particular antigen or epitope refer to binding that is measurably different from non-specific interactions. Specific binding can be measured by determining binding of a molecule compared to binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. Specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or epitope is 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 Specific binding to a particular antigen or epitope can be demonstrated by an antibody having a KD for the antigen or epitope of M or greater. Here, KD refers to the dissociation rate of a particular antibody-antigen interaction. In some embodiments, a multispecific antibody that specifically binds to an antigen has a KD for the antigen or epitope that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, or 10,000-fold or greater compared to a control molecule. Specific binding to a particular antigen or epitope can also be demonstrated by an antibody having a K A or K B for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, or 10,000-fold greater than the control. K A or K B refers to the association rate of a particular antibody-antigen interaction.

[0086] The term "staple" refers to two domains covalently linked. In one embodiment, the two domains may be covalently linked via at least one disulfide bond. For example, an scFv domain having at least one disulfide bond linking the VH and VL is called a stapled scFv. An Fab region having at least one disulfide bond linking the light chain portion and the heavy chain is called a stapled Fab.

[0087] Example The following examples are illustrative only and not limiting of the present invention. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially the same or similar results.

[0088] Example 1: Staple Binding Domain and Stability of 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 form a quadruple binding specificity, while the light chain may remain unmodified. In penta-GNC antibodies, one scFv domain is added to either the N- or C-terminus of the light chain to obtain a quintuple binding specificity (Figure 1 and Table 1). When an scFv domain is attached to each of the N- and C-termini of the light chain, the antibody obtains a fifth and sixth binding specificity, becoming a hexa-GNC antibody. Modifications 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, VFv fragments and scFv domains are added to each Fv fragment and scFv domain. L 100 and V H 44 (Kabat) provides a method for introducing disulfide bonds, i.e., stapling. L and V H The disulfide bond between can be applied to all scFv domains to stabilize the overall structure, or the disulfide bond can be introduced at any position into at least one selected scFv domain.

[0089] To analyze the effect of stapled scFv domains, a pair of penta-GNC antibodies (SI-1P1 and SI-1P2) with identical binding specificities (SEQ ID NOs: 1-4 and 5-8, respectively) was created. Based on the numbering system in Figure 1, the heavy chains of these two antibodies are αCD3 scFv in D1 and αEGFR V in D2. H (Fv-CH1-Fc configuration), αPD-L1 at D3, α4-1BB at D4, and αEGFR V at D5. L SI-1P2 (but not SI-1P1) has "stapled" scFv domains at D1, D3, D4, and D5, i.e., αCD3 scFv [V] at D1 of its heavy chain, as shown in Table 1. 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 100G->C] and its light chain contains αHER3 scFv [V H 44G->CV L 100G->C] (Kabat numbering).

[0090] Both SI-1P1 and SI-1P2 were cloned into the vector pTT5 using a modular cloning strategy using the restriction sites HindIII / SalI / NheI / BamHI / BspEI / PacI. These pentaGNC antibody constructs were expressed at acceptable titers for 5 and 9 days using both HEK and ExpiCHO expression systems, respectively, and purified on a 5 mL MabSelect Protein A column followed by size exclusion using a hiload 16 / 600 200 pg preparative SEC column on either an Äkta Avant or Purifier system. SEC aggregation was analyzed using a Waters HPLC coupled to a multi-angle light scattering detector (MALS, Wyatt Systems), and accurate molecular weights were determined by dn / dc calculations. In subsequent analyses, the melting temperatures of the resulting proteins were determined using a dynamic light scattering instrument (Wyatt Systems). As can be seen from all analyses performed, as shown in Table 2, the disulfide-linked (i.e., "stapled") pentaGNC antibody exhibited greater stability.

[0091] Antibody-based proteins are most often purified by Protein A affinity chromatography. Protein A resins bind to the C of the Fc domain. H2 -C H3 Protein A captures antibodies at the binding sites at the interface. H3 Family Fv V H It also binds to the V domain. H For most antibody-based platforms, this is not an issue, as the domain is usually located in the heavy chain. H3 When an scFv containing V binds to a light chain, H The V domain can be bound to Protein A resin during purification, whereby light chain monomers and dimers contaminate the heavy-light chain heterotetramer. H3 A potential obstacle in generating multispecific antibodies containing domains is the presence of additional contaminants in the Protein A eluate, which can be particularly problematic if the light chains are expressed more efficiently than the heavy chains, and large amounts of light chain contaminants are co-purified with the desired protein assembly.

[0092] To adequately disrupt Protein A binding of VH3 family members, a structural approach was employed to block the binding interface. The crystal structure 1DEE (Graille M. et al. Proc. Nat. Acad. Sci. 2000) of V H3 This indicates that residue R19 (Kabat numbering) in S19 directly contacts both chains of protein A domain D. In particular, removing contacts with Q32 and D36 significantly weakens the interaction. Therefore, R19 was mutated to serine, because the side chain of serine is too short to form these interactions. Furthermore, because S19 naturally occurs in other VH family members, this suggests that it may be less immunogenic than other substitutions.

[0093] Mutation R19S (Kabat numbering) was added to the V H3 The R19S mutation was engineered into the FR1 region of the VH domain for the scFv containing the R19S mutation. Specifically, the penta-GNC antibody SI-77P1 (SEQ ID NOs: 41-44) has an R19S mutation in the light chain sequence encoding the anti-CD19 scFv in domain 6, while the hexa-GNC antibodies SI-55-H11 (SEQ ID NOs: 53-56), SI-55H12 (SEQ ID NOs: 57-60), SI-77H4 (SEQ ID NOs: 61-64), and SI-77H5 (SEQ ID NOs: 65-68) have an R19S mutation in the light chain sequence encoding the anti-HER3 scFv domain in domain D5 and the anti-CD19 scFv in domain 6. Because the target residue is located in the Protein A binding interface (4), the R-to-S mutation disrupts the interaction with Protein A. Eliminating Protein A binding to the light chain scFv prevents light chain monomers and dimers from binding to Protein A during purification. This results in a more homogeneous product free of light chain contaminants. Up to two V per light chain H3 In the case of hexaGNC, which may contain scFv, this mutation is crucial for efficient purification of the desired product.

[0094] Wild-type IgG1 antibodies contain an activated Fc domain that binds to Fcγ receptors on immune cells and C1q, the first component of the complement cascade. These binding capabilities allow antibodies with activated Fc to induce effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity against antigen-bearing cells. However, in the context of T cell redirection, activated Fc domains can exacerbate cytokine release syndrome and cause off-target cytotoxicity (Strohl & Naso, Antibodies, 2019). Therefore, null Fc domains incorporating silencing mutations that weaken binding to Fcγ receptors and complement can enhance the efficacy of T cell-redirecting 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 interactions with Fcγ receptors or C1q and reduce Fc effector function (Saunders, Frontiers Immunol. 2019). Among them, the L234A, L235A, and G237A mutations reduce ADCC and ADCP by reducing binding to Fcγ receptors. The K322A mutation reduces binding to C1q and eliminates CDC. Furthermore, the N297A mutation removes the Fc glycosylation site, resulting in an aglycosylated Fc domain that does not interact as strongly with receptors.

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

[0096] Example 2: Optimization of the binding domain While the usefulness of multispecific GNC antibodies is determined by selecting binding specificities, optimizing commonly used binding domains can improve antibody efficacy. PentaGNC and hexaGNC antibodies (collectively referred to as GNC antibodies, as shown in Table 1) were cloned, expressed, and produced using materials and methods similar to 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 No. PCT / US2018 / 039143; incorporated herein in its entirety) was synthesized as a scFv (V H -V L or V L -V H The sequences were used as 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 Fab region of the heavy chain monomer (orientation) or the Fab region of the heavy chain monomer. Another anti-CD3 variable domain sequence, 283E3, was identified, cloned, and humanized as 283E3 H1 (SEQ ID NOS: 101-104), which was used to encode the Fab region in D2 of the heavy chain monomer of each of the seven Penta-GNC antibodies (Table 1).

[0098] The anti-PD-L1 variable domain sequence PL221G5 (Applicant's application no. PCT / US2018 / 039144; incorporated herein in its entirety) was humanized and used to encode either the "non-stapled" or "stapled" (SEQ ID NOs: 105-108) scFv domains located in D3 of the heavy chain monomer.

[0099] The anti-4-1BB variable domain sequence 466F6 (Applicant's application no. PCT / US2018 / 039155; incorporated herein in its entirety) was humanized and used to encode either the "non-stapled" or "stapled" (SEQ ID NOs: 109-112) scFv domains in D4 of the heavy chain monomer.

[0100] Example 3: Octet analysis of binding affinity To evaluate the functionality of the GNC antibody, we evaluated the binding affinity of each domain of the PentaGNC antibody by Biolayer Interferometry (Octet384 system). The PentaGNC antibody was captured onto a probe using anti-human Fc (AHC chip) to generate individual epitopes (CD3ε / δ) and EGFR. 4-1BB and PD-L1 (Acro Biosystems) were used as analytes to perform a kinetic analysis (K). off / K on ) to determine the dissociation constant (K D ) were determined. As shown in Table 3, the binding constants of the individual domains of either SI-1P1 or SI-1P2 were all within the reported ranges, predetermining the single affinity of the monoclonal antibody or scFv molecule.

[0101] Octet analysis confirmed that the GNC antibodies maintained binding to all of their cognate antigens. The GNC antibodies were loaded onto the AHC sensor at 10 μg / ml for 180 seconds. This was followed by a 60-second baseline step, a 180-second association step with 100 nM of commercial human antigen, and a 360-second dissociation step. For 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 fits and extract the affinity KD values ​​reported in Table 4. The data demonstrate that each binding domain retains its binding affinity even when placed at different positions on the GNC antibody.

[0102] Example 4: T cell activation To verify the functionality of the GNC antibody, we evaluated T cell activation of the penta-GNC antibody. Using a T cell activation assay, we compared the potency of SI-1P1, which binds both EGFR and HER3, with that of an EGFR-tetra-GNC antibody (which binds to EGFR but not HER3), an FITC-tetraspecific antibody (which binds neither EGFR nor HER3), and a bispecific antibody (which binds only to EGFR and HER3). Human pancreatic cancer cells (BxPC3), which express high levels of EGFR and low levels of HER3, were used as target cells (Table 5). BxPC3 cells were treated with a dissociation reagent (TrypLE Express) and then seeded in quadruplicate into 384-well plates at a density of 1500 cells / well using a BioTek EL406 and allowed to adhere for 24 hours. Jurkat CD3 NFAT effector cells (Jurkat Lucia cells, Invivogen) were then added at a 5:1 cell ratio, and the GNC antibody was added in a 10-point, 10-fold serial dilution ranging from 50 nM to 0.5 fM and incubated for 4 hours. The reading was obtained by adding Promega Bright-Glo reagent, and luminescence was measured using a Clariostar Plus microplate reader (BMG-Labtech). Data were plotted on a logarithmic scale using Graphpad Prism and fitted to a nonlinear variable slope equation (Figure 2). The data show that the penta-GNC antibody (SI-1P1) exerts similar potency (SI-1P1 = 1.456 pM, EGFR-tetra-GNC = 1.028 pM, FITC-tetra-GNC = 13.41 pM) 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 TDCC mediated by the GNC antibody, we used SI-1P1 (a penta-GNC antibody capable of binding to the tumor antigens EGFR and HER3) and compared it with control antibodies. The control antibodies included an EGFR-tetra-GNC antibody that binds only to EGFR, a FITC-tetraspecific antibody that binds neither to EGFR nor HER3, and a control bispecific antibody that binds only to the tumor antigens EGFR and HER3 (Table 6). Serial dilutions (0 to 30 nM; dilutions 1 to 5) of antibodies were added in a total volume of 50 μl to white 384-well plates containing luciferase-treated MDA-MB-231 or HeLa cells (both with high EGFR and low HER3 (Table 5)) seeded 24 hours prior and grown at 37°C and activated T cells (seeded immediately before drug administration; effector:target ratio 5:1). After an additional 72 hours, 20 μl of Bright-Glo (Promega) was added to the wells, and the luminescence versus viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. The data were fitted to a sigmoidal function to calculate EC50 values ​​(Figure 3). For MDA-MB-231 cells shown in Figure 3A, the EC50 values ​​were 0.01575 pM (SI-1P1), 0.01646 pM (EGFR-tetra-GNC control antibody), and 1.882 pM (FITC-tetraspecific control antibody). For cells shown in Figure 3B, the EC50 values ​​were 1.161 pM (SI-1P1), 1.635 pM (EGFR-tetra-GNC control antibody), 3736200 pM (FITC-tetraspecific antibody), and 4500 pM (bispecific control antibody). The data show that with an increased number of binding specificities, the PentaGNC antibody exerts higher TDCC potency compared to a control antibody with fewer binding specificities.

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

[0105] Both SI-49P3 and SI-49P1 can recognize a single tumor antigen via their Fab region, while also extending their binding specificity to CD3, PD-L1, 4-1BB, and NKG2D. To demonstrate that SI-49P3 retains its T cell activation potential, BxPC3 cells were treated with a dissociation reagent (TrypLE Express) and then seeded in quadruplicates into 384-well plates at a density of 1500 cells / well using a BioTek EL406. They were then allowed to adhere for 24 hours. Jurkat CD3 NFAT effector cells (Jurkat Lucia cells, Invivogen) were then added at a 5:1 cell ratio, and GNC reagent was added in a 10-point, 10-fold serial dilution from 50 nM to 0.5 fM. The cells were then incubated for 4 hours. Promega Bright-Glo reagent was added, and luminescence was measured using a Clariostar Plus microplate reader (BMG-Labtech). Data were plotted on a logarithmic scale and fitted to a nonlinear variable slope equation using Graphpad Prism (Figure 4A). The data demonstrate that the addition of NKG2D receptors does not affect T cell activation, and that the Penta-GNC antibody can induce effective T cell activation (EC50 = 88.1 pM) and engage T cell antigens to target tumor cells.

[0106] The TDCC of the NKG2D class of the penta-GNC antibody was evaluated using SI-49P1. Control antibodies included a tri-GNC antibody lacking binding specificity for both PD-L1 and 4-1BB, and a tetra-GNC antibody lacking the NKG2D receptor. Serially diluted (0 to 30 nM; dilution factors 1 to 5) GNC protein was added in a total volume of 50 μl to a white 384-well plate containing luciferase-treated MDA-MB-231 cells (expressing MICA and mesothelin; seeded 24 h prior and grown at 37°C) and activated T cells (seeded immediately before drug administration; effector:target ratio 5:1). After an additional 72 h, 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 data were fitted to a sigmoidal function to calculate EC50 values, which were 0.1865 pM (SI-49P1), 0.3433 pM (Tri-GNC control antibody), and 5.356 pM (Tetra-GNC control antibody) (Figure 4B). The data demonstrate that adding the NKG2D receptor to the Tetra-GNC antibody improves TDCC potency, and adding the αPD-L1 and α4-1BB domains to the Tri-GNC antibody, which has binding specificity for NKG2D, significantly improves TDCC potency. In other words, GNC antibodies can accommodate multiple binding specificities to modulate, coordinate, and induce optimized immune responses against target cells (e.g., cancer).

[0107] Example 7: NKG2D receptor at D2 of antibody-like NKG2 proteins To test the utility of antibody-like NKG2D proteins with a native receptor at D2, the sequence for human NKG2D (residues F78 to V216) was cloned and inserted into the V at D2 region in the context of an expression plasmid encoding SI-49P10 (αCD3xNKG2DxαPD-L1xα4-1BBxαEGFR, SEQ ID NOs: 117-200). H and V LThe domains were replaced. SI-49P10 was expressed and purified with Protein A according to the materials and methods described above, and showed very low aggregation (analytical SEC: 95.64% of the target peak), indicating that antibody-like GNC proteins with non-antibody binding moieties (e.g., NKG2D receptors) in the D2 region of the heavy chain are likely to be highly stable. The penta-GNC proteins SI-49P6 (αCD3xNKG2DxαPD-L1xα4-1BBxαCD19, SEQ ID NOs: 123-126) and SI-49P7 (αCD3xNKG2DxαPD-L1x41BBL trimerxαCD19, SEQ ID NOs: 127-130) were cloned, expressed, and purified in the same manner as described above.

[0108] To ensure that the NKG2D dimer retained full functionality, Octet binding to human MICA was assessed. SI-49P10 was loaded onto an AHC chip and bound to His-tagged MICA. The extracted KD values ​​confirmed that NKG2D retained binding activity when located at position D2 (Table 4). SI-49P10 exhibited a KD D The value was 1.84 nM. In comparison, SI-49P3 (NKG2D dimer at D5) had a similar K D The K value was 1.39 nM. Other domains of SI-49P10 also retained high binding affinity to their cognate antigens (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 with serial dilutions (0 to 100 nM) of the analyte GNC protein. The resulting K D The K values ​​were 7.763 nM (SI-49P6) and 10.67 nM (SI-49P7), further demonstrating that receptor proteins at the D2 position retain target binding (Table 4). These K values ​​with antigen-loaded ligands DThe affinity values ​​were slightly lower compared to experiments in which GNC proteins were loaded, which may be due to the inactive conformation of the MICA protein when loaded as a ligand or an incompletely exposed epitope. Nevertheless, the potent femtomolar (<1 pM) TDCC against MICA-bearing MDA-MB-231 cells induced by these proteins with NKG2D at the D2 position (Figure 8) indicates that the NKG2D receptor retains active binding at the D2 position. This makes the GNC platform highly adaptable to the placement of each domain.

[0109] Example 8: 4-1BB Ligand as Binding Domain 4-1BB is a costimulatory immune checkpoint TNFR receptor expressed by activated T cells and NK cells. 4-1BB ligand or CD8 +Activation by agonist antibodies on T cells leads to increased proliferation, cytokine production, and survival. To optimize 4-1BB-mediated immune responses, we evaluated the functionality of different domains 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 (referred to as 4-1BB effector cells) driven by a response element that can respond to 4-1BB ligand / agonist antibody stimulation. 4-1BB effector cells are cultured in RPMI-1640 containing 10% FBS. Prior to the assay, these cells are counted and replated at 500 cells / well in a 384-well plate (Corning 3570). The 96-well dilution block was robotically stamped into a 384-well quadrant (Opentrons OT-2 liquid handling robot), and test article experiments were performed in quadruplicate. 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. Briefly, after adding 20 μL to the 4-1BB assay plate and incubating for approximately 15 minutes, the resulting luminescence was measured using a BMG Clariostar plate reader. The activation curve was analyzed and generated using GraphPad software as a 4PL curve (Figure 5). The results show that the 4-1BB ligand trimer (4-1BBL trimer, SEQ ID NOs: 113-114) induces stronger activation of 4-1BB signaling compared to the monomeric 4-1BB ligand, monomeric Fc, and anti-4-1BB scFv. Penta-GNC antibodies SI-55P4, SI-55P10, and SI-79P3 (sequence numbers 29-32, 37-40, and 49-52, respectively) were constructed to have the 4-1BBL trimer as the binding domain in D4 (Table 1 and Figure 7).

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

[0111] Example 9: Humanized EGFR binding domains Cetuximab is a chimeric mouse / human monoclonal antibody used to treat EGFR-expressing metastatic colorectal cancer, non-small cell lung cancer, and head and neck cancer. Humanized antibodies can be obtained. In this example, humanized sequences encoding anti-EGFR binding sites (H1, H4, H7, and H7-staple) (SEQ ID NOs: 69-72, 73-76, 77-80, and 81-84, respectively) were cloned into expression cassettes to produce 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) (SEQ ID NOs: 25-28, 29-32, 45-48, 49-52, and 33-36, respectively) (Table 7). Each expression cassette was transfected into 25 mL of ExpiCHO cells and expressed for 8 days. Each pentaGNC antibody was 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, we verified that each pentaGNC antibody containing the humanized anti-EGFR domains H1, H4, or H7 could bind to human EGFR (Figure 6 and Table 7). Each pentaGNC antibody was loaded at 10 μg / ml on 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 the pentaGNC antibody binds to EGFR with affinity in the low nanomolar range (Table 7).

[0112] To produce hexaGNC antibodies, the humanized anti-EGFR domain H7 (SEQ ID NOs: 77-80) was cloned into an expression cassette to produce anti-EGFR hexaGNC antibodies. The humanized binding domain was placed in the Fab or scFv at D1 in the hexaGNC antibodies SI-77H4 (SEQ ID NOs: 61-64) and SI-55H11 (SEQ ID NOs: 53-56). The control antibody SI-77H5 (SEQ ID NOs: 65-68) contains the anti-EGFR-binding Fab region encoded by the cetuximab mouse. The expression cassette was transfected into 25 mL of ExpiCHO cells and expressed for 8 days. Each hexaGNC antibody was then collected and purified by Protein A affinity chromatography. HexaGNC antibodies with good titers were obtained (Table 8). Analytical SEC data after Protein A purification demonstrated that each hexaGNC antibody containing the humanized anti-EGFR domain can be expressed with low aggregation (Table 8). Using Octet, we verified that each of these hexaGNC antibodies containing a humanized anti-EGFR domain can bind to human EGFR (Tables 4 and 8). The hexaGNC antibodies were loaded at 10 μg / ml on 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 demonstrates that each hexaGNC antibody binds to EGFR with affinity in the low nanomolar range (Table 8).

[0113] Example 10: Humanized CD19 binding domains CD19 is a biomarker for B lymphocyte development and lymphoma diagnosis. CD19-targeted therapies based on CD19-specific chimeric antigen receptor-expressing T cells (CAR-T) have been shown to enhance CD19 expression due to their antitumor capabilities. + It has been used in patients with lymphoma and leukemia (e.g., non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphocytic leukemia). Humanized CD19 binding domains are preferred herein.

[0114] All computational steps were performed using the Discovery Studio package (Dassault Systèmes). First, a structural model was generated using the mouse BU12 sequence. Antibody framework regions in the input sequence were identified using a hidden Markov model (HMM) and aligned to a database of antibody variable domains. Models were constructed and cored using this alignment using the MODELLER software. CDR loop modeling was performed by structurally mapping the CDRL1, CDRL2, CDRL3, CDRH1, and CDRH2 regions to known canonical classes, and loop models were constructed similarly to the framework. Framework regions from the mouse BU12 antibody were aligned and matched to the closest human germline sequence. Excluding critical structural residues, the CDR regions were copied to the human sequence (Vernier residues [Almagro and Fransson, 2008]). Mutations predicted to stabilize the previously constructed structural model were evaluated using 1000 steps of steepest descent with an RMS gradient tolerance of 3, followed by conjugate gradient minimization to select stabilizing mutations matching frequent human residues based on the individual and combined -ΔΔG versus the initial model. The resulting final sequences were tested for humanization using the Abysis web server according to the method of Abhinandan and Martin (2007).

[0115] To confer binding specificity to penta- or hexa-GNC antibodies for targeting B-cell malignancies, the sequences encoding anti-CD19V L and V H The sequence encoding the domain is designated as modified V L SEQ ID NOs: 87, 88, 121, 122, 131, 132 and modified V L and R19S mutation-containing V HThe anti-CD19 scFv domain was formed by selecting from SEQ ID NOS: 85, 86, 87, and 88 (R19S mutations in Table 1 and Example 1) and ligating them with a (G4S)x4 linker. The corresponding gene sequences were cloned into different positions of the penta- or hexa-GNC antibody and digested with restriction enzymes into the pTT5 expression plasmid to obtain the appropriate heavy or light chain moiety. As described above, the anti-CD19 penta-GNC and hexa-GNA antibodies shown in Table 1 were produced and characterized. Octet analysis of CD19 binding affinity showed that each GNC antibody retained CD19 binding affinity within the expected range when placed in the light chain portion of the GNC antibody monomer (Table 4).

[0116] Example 11: Penta GNC antibody with optimized binding domain The PentaGNC antibody was evaluated in a TDCC assay due to its optimized specific binding to 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 the same binding specificity. SI-55P9 has a humanized anti-EGFR binding domain, and SI-55P10 uses a 4-1BBL trimer to activate 4-1BB signaling via the anti-4-1BB binding domain in SI-55P9. To assess the impact of these differences in the TDCC assay, serial dilutions (0 to 30 nM; 1 to 5 dilutions) of the penta-GNC proteins SI-1P1, SI-55P9, and SI-55P10 were added to a white 384-well plate containing luciferase-treated BxPC3 (high EGFR expression) cells (seeded 24 h prior and grown at 37 °C) and activated T cells (seeded immediately prior to drug administration; effector:target ratio = 5:1 (SI-1P1) and 7:1 (SI-55P9 and SI-55P10)) in a total volume of 50 μL. After an additional 72 h, 20 μL of Bright-Glo (Promega) was added to the wells, and luminescence relative to the viability of the luciferase-treated tumor cells was determined using a Clariostar plate reader. As shown in Figure 7, the potencies of SI-55P9 and SI-55P10 were similar to each other compared to SI-1P1. The data were fitted to a sigmoidal function to calculate EC50 values, which were 0.2814 pM (SI-1P1), 0.4871 pM (SI-55P9), and 0.7358 pM (SI-55P10), all in the picomolar range. These results demonstrate that the composition of binding specificities determines the potency of the PentaGNC antibody in the TDCC assay, and further optimization of each binding domain can be evaluated using other in vitro and in vivo assays.

[0117] Example 12: Redirected T cell cytotoxicity Any antibody-based binding domain can be converted into Fab or scFv format and inserted directly into the GNC antibody. For example, the GNC antibody features the addition of a fifth and / or sixth binding domain to the light chain portion. If the binding specificity on the heavy chain can be dedicated to frequently used targets such as CD3, PD-L1, and 4-1BB, the utility of the GNC platform can be flexible in terms of selecting target tumor antigens and pairing the less flexible heavy chain with the desired light chain portion. Herein, three tetra-GNC antibodies were selected (Applicant's Application No. PCT / US2019 / 024105; incorporated herein in its entirety) and evaluated using an in vitro redirected T cell cytotoxicity (RTCC) assay and an in vivo human tumor xenograft model.

[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 a 4-day period was determined by live-cell imaging of cultures containing PBMCs (single donor) and red fluorescently labeled tumor cells. PBMCs (50,000 cells / mL) were used at a 4:1 (PBMC:A549) ratio to challenge NucRed-transduced A549 lung adenocarcinoma cells. Assay wells were run in triplicate with 1 nM SI-35E20 or GNC-free (buffer only) as a negative control, and target cell proliferation was monitored for 94 hours. The data demonstrate that SI-35E20 can inhibit target cancer cell proliferation 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 (a human leukemia cell line) as target cells and PBMCs from one donor as effector cells. RTCC assays were performed at an E:T ratio of 1. At 100 pM, SI-38E17-mediated RTCC was tracked over 48 hours using an IncuCyte™ system to detect target cell proliferation. SI-38E17 mediated potent RTCC functional activity against Nalm-6 (Figure 10). The data obtained demonstrate that tetra-GNC antibodies, such as SI-38E17, can inhibit target cancer cell proliferation over time.

[0120] SI-39E18 is a tetra-GNC antibody capable of binding to CD3, EGFRvIII, PD-L1, and 4-1BB. RTCC assays confirmed greater cell killing than the solvent control, as shown in Figure 11. Figure 11 shows average red fluorescence intensity measurements over time for three different PBMC donors. Target cell numbers increased in the presence of SI-39E18 or buffer control, as measured by fluorescence intensity over the first 24 hours of culture. Here, effector cells were preincubated with SI-39E18 or control for three days before adding target cells. Between 24 and 48 hours after adding target cells to the culture, target cell numbers began to decrease in the presence of SI-39E18 stimulation, whereas they did not decrease in wells containing buffer control. The more modest decrease in target cells in the buffer control samples in both conditions after 48 hours is likely due to nutrient depletion or an allogeneic T cell response to MHC-mismatched target cells. The data obtained demonstrate that tetra-GNC antibodies, such as SI-39E18, can prevent target cell proliferation over time.

[0121] Example 13: Human tumor xenograft model The ability of SI-38E17 to slow tumor growth in vivo was examined in a mouse xenograft model (Figure 12). Human B-cell leukemia cells (JVM-3) were cultured at 5x10 6 Cells / mouse were subcutaneously implanted into the right flank of NCG mice, and tumor volumes were 50-80 mm 3 Donor PBMCs were cultured at 2 x 10 7 SI-38E17 cells / mouse were intraperitoneally injected. Each group consisted of five 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 vehicle group was 1298 mm 3 All three doses of SI-38E17 showed significant tumor-suppressing effects, with the mid-dose of 0.005 mg (drug:TCR = 12.5:1) showing the best tumor suppression (TGI = 84%). All mice in the vehicle control group died before day 22, while one mouse in the low-dose group, three in the mid-dose group, and one in the high-dose group had tumor-free before day 40. Thus, tetra-GNC antibodies, such as SI-38E17, demonstrate potent tumor-suppressing effects in vivo across multiple doses.

[0122] The ability of SI-39E18 to slow tumor growth in vivo was examined in a mouse xenograft model (Figure 13). NCG mice were injected with 5x10 6 UM-UC-3-EGFR VIII cells derived from human bladder cancer were inoculated. The average tumor volume was 50-80 mm. 3 When it reaches 5×10 6Human PBMCs were injected intraperitoneally per mouse (100 μl), and different doses of SI-39E18 were administered intravenously. Each group consisted of five animals, and the indicated doses were administered intravenously once daily for a total of 18 doses. The first day of administration was defined as D1. Tumor growth after SI-39E18 administration is shown in the figure, demonstrating that SI-39E18 induces strong tumor growth inhibition across multiple doses. On the day of discontinuation (D18), tumor volume remained zero for three consecutive days in all dose groups (low dose group 0.001 mg, medium dose group 0.01 mg, high dose group 0.1 mg), while the volume significantly increased in the vehicle group. Thus, tetra-GNC antibodies, such as SI-39E18, demonstrate strong in vivo biological activity across multiple doses.

[0123] Example 14: More complete cytotoxicity A hexa-GNC antibody was generated as a single-antibody therapeutic to investigate its multifunctionality. SI-55H11 (SEQ ID NOs: 53-56) is a hexa-GNC antibody with binding specificity for CD3 (D1), EGFR (D2), PD-L1 (D3), and 4-1BB (D4) on the heavy chain monomer, and HER3 (D5) and CD19 (D6) on the light chain monomer (Table 1). TDCC assays were used to determine the effect of targeting PD-L1 and 4-1BB on T cell-mediated tumor cell killing compared with a trispecific antibody targeting CD3 (D1) on T cells and both EGFR (D2) and HER3 (D5) on tumor cells (Table 4). Serial dilutions (0 to 30 nM; dilution factors 1 to 5) of Tri- or Hexa-GNC were added in a total volume of 50 μl to white 384-well plates containing luciferase-treated BxPC3 (high EGFR-expressing) cells (seeded 24 h prior and grown at 37°C) and activated T cells (seeded immediately before drug administration; effector:target ratio = 5:1). After an additional 72 h, 20 μl of Bright-Glo (Promega) was added to the wells, and luminescence versus viability of luciferase-treated tumor cells was determined using a Clariostar plate reader. The data were fitted to a sigmoidal function to calculate EC50 values ​​and maximum killing (Figure 14). The data show that the Hexa-GNC antibody exerts more complete killing compared to the trispecific antibody (bottom plateau = 46.92% viability; SI-55H11 bottom plateau = 2.992% viability). Data suggest that activating T cells with CD3 and simultaneously targeting immune regulatory proteins, such as PD-L1 immune checkpoints, and activating 4-1BB is an effective combination strategy to induce a GNSS response of the immune system against target cells, leading to more complete tumor depletion.

[0124] Example 15: Targeting Multiple Tumor Antigens As shown in Examples 12 and 13, attempts can be made to anchor immune targets to the heavy chain. Binding domains on the light chain can be dedicated to tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), and neoantigens. Herein, several hexa-GNC antibodies were generated and subjected to TDCC assays. GNC antibodies were evaluated to determine whether additional tumor targeting specificity could enhance T cell-mediated tumor cell killing (Figure 15). Herein, SI-55P9 (SEQ ID NOs: 33-36), a penta-GNC antibody capable of binding to EGFR, CD3, PD-L1, and 4-1BB via its heavy chain monomer and CD19 via its light chain monomer, was compared with SI-55H11 (SEQ ID NOs: 53-56), a hexa-GNC antibody with the same binding specificity but adding a sixth specificity to HER3 via its light chain monomer. Serial dilutions (0 to 30 nM; dilution factors 1 to 5) of each GNC antibody were added to a white 384-well plate containing 500 luciferase-treated BxPC3 (high EGFR; low HER3) cells (seeded 24 h prior and grown at 37°C) and 2500 activated T cells (seeded immediately prior to drug administration; effector:target ratio 5:1) in a total volume of 50 μl. After an additional 72 h, 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. EC50 values ​​were calculated by fitting the data to a sigmoidal function. The data show that the hexaGNC antibody has higher potency in the TDCC assay (SI-55P9 EC50 = 0.5727 pM; SI-55H11 EC50 = 0.09387 pM) compared to the parent pentaGNC antibody, which lacks one of the tumor-targeting domains. Thus, the additional anti-tumor antigen-binding domain can improve the biological function of the GNC antibody, even against cells expressing low levels of a TAA (in this case, HER3).

[0125] The above description and examples provide a complete description of the structure and use of exemplary embodiments. While certain embodiments have been described above with a certain degree of particularity or with 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 present invention. For example, while the above examples may include binding domains in specific locations, these are provided for comparison purposes only and not as limitations. It is specifically contemplated herein that any combination of binding domain configurations and their locations on the GNC protein may be used. Therefore, the exemplary embodiments of the present invention are not intended to be limited to the specific embodiments disclosed. Rather, they include all modifications and alternatives that fall within the scope of the disclosure. Furthermore, where appropriate, any aspect of the above examples can be combined with any aspect of other described examples to form additional examples having similar or different characteristics and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or to several embodiments.

[0126] [Table 1]

[0127] a 284A10, see applicant's application number PCT / US2018 / 039143 b 284A10 H1 (SEQ ID NOs: 93-100), humanized anti-CD3 variable domain sequences encoding scFv domains or Fab regions in "unstaped" or "stapled" form (SEQ ID NOs: 155-108) c 283E3 H1 (SEQ ID NOs: 101-104), 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 the R19S mutation

[0129] e SI-huBU12 H1 VH (SEQ ID NOs: 85, 86) and SI-huBU12 H1 VL (SEQ ID NOs: 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 the R19S mutation

[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 Listing JPEG2025037909000009.jpg134101JPEG2025037909000010.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 TSGMGVG 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. 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; a second binding domain (D2) comprising a light chain portion; An Fc region, A third binding domain (D3); and a fourth binding domain (D4) located at the C-terminus; Including, the light chain portion comprises a fifth binding domain (D5) covalently attached to the C-terminus and / or a sixth binding domain (D6) covalently attached to the N-terminus; D1, D2, D3, D4, D5 and D6 are multispecific antibody-like proteins each having binding specificity for a tumor antigen, an immune signaling antigen or a combination thereof.

2. The multispecific antibody-like protein of claim 1 , wherein D2 comprises a dimer that binds to CL and CH1.

3. The multispecific antibody-like protein of claim 1 , wherein D2 comprises a Fab region.

4. The multispecific antibody-like protein of claim 1 , wherein D2 comprises a receptor.

5. The multispecific antibody-like protein of claim 1 , wherein the D2 comprises NKG2D.

6. the light chain portion comprises a fifth binding domain (D5) covalently linked to the C-terminus, The multispecific antibody-like protein of claim 1 , wherein the multispecific antibody-like protein is pentaspecific.

7. the light chain portion comprises a sixth binding domain (D6) covalently linked to the N-terminus; The multispecific antibody-like protein of claim 1 , wherein the multispecific antibody-like protein is pentaspecific.

8. the light chain portion comprises a fifth binding domain (D5) covalently attached to the C-terminus and a sixth binding domain (D6) covalently attached to the N-terminus; The multispecific antibody-like protein of claim 1 , wherein the multispecific antibody-like protein is hexaspecific.

9. The multispecific antibody-like protein of claim 1 , wherein D1, D2, D3, D4, D5 and D6 are each independently an scFv domain, a receptor or a ligand.

10. The multispecific antibody-like protein of claim 1 , wherein D2 has binding specificity for CD3 or a tumor-associated antigen (TAA).

11. 2. The multispecific antibody-like protein of claim 1, wherein D1, D2, D3, D4, D5 and D6 each independently have binding specificity for an antigen selected from a receptor on a T cell, an immune checkpoint receptor, a costimulatory receptor, a receptor on a lymphocyte or myeloid cell, a tumor-associated antigen (TAA), a tissue antigen, a neoantigen, a tumor-specific antigen (TSA), a glycoprotein, and combinations thereof.

12. The multispecific antibody-like protein of claim 11 , wherein the binding domain for a receptor on the T cell is adjacent to the binding domain of the tumor-associated antigen (TAA).

13. The multispecific antibody-like protein of claim 11 , wherein the binding domain for a receptor on a T cell is adjacent to a binding domain for a receptor on a lymphocyte or myeloid cell.

14. The multispecific antibody-like protein of claim 11 , wherein the receptor on the T cell comprises CD3, a T cell receptor, or a complex thereof.

15. The multispecific antibody-like protein of claim 11, wherein the immune checkpoint receptor comprises PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, CD160, LOX-1, siglec-15, CD47, SIRPα, or a combination thereof.

16. The multispecific antibody-like protein of claim 11, wherein the costimulatory receptor comprises 4-1BB, CD28, OX40, GITR, CD40, ICOS, CD27, CD30, CD226, or a combination thereof.

17. 15. The multispecific antibody-like protein of claim 14, wherein the tumor associated antigen (TAA) comprises 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.

18. The multispecific antibody-like protein of claim 1, wherein D1 has binding specificity for CD3, CD20, EGFR or a derivative thereof.

19. The multispecific antibody-like protein of claim 1 , wherein said D2 has binding specificity for EGFR, CD3, HER2, MSLN, NKG2D ligand or derivatives thereof.

20. The multispecific antibody-like protein of claim 1, wherein the D3 has binding specificity for PD-L1.

21. The multispecific antibody-like protein of claim 1, wherein the D4 comprises a 4-1BBL trimer or has binding specificity for 4-1BB or a derivative thereof.

22. The multispecific antibody-like protein of claim 1 , wherein said D5 has binding specificity for HER3, CD19, NKG2D ligand or derivatives thereof.

23. The multispecific antibody-like protein of claim 1 , wherein D6 has binding specificity for CD19.

24. The D1, D3, D4, D5 or D6 is G ｘ S ｙ ) ｎ 2. The multispecific antibody-like protein of claim 1 , comprising a linker, wherein 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.

25. A guidance and navigation control protein comprising a dimer of the multispecific antibody-like protein of claim 1.

26. An isolated nucleic acid sequence encoding the amino acid sequence of the multispecific antibody-like protein of claim 1.

27. 27. An expression vector comprising the isolated nucleic acid sequence of claim 26.

28. 27. A host cell comprising the isolated nucleic acid sequence of claim 26, The host cell is a prokaryotic or eukaryotic host cell.

29. Culturing a host cell comprising the isolated nucleic acid sequence such that the DNA sequence encoding the multispecific antibody-like protein of claim 1 is expressed; purifying the multispecific antibody-like protein; A method for producing a multispecific antibody or monomer, comprising the steps of:

30. 1. A method for treating or preventing cancer, an autoimmune disease, or an infectious disease, comprising:

30. A method comprising administering a pharmaceutical composition comprising the purified multispecific antibody of claim 29 or the multispecific antibody-like protein of claim 1.

31. 30. A multispecific antibody comprising a cytotoxic agent or an imaging agent attached via a linker to the multispecific antibody of claim 29, The immunoconjugate, wherein the linker comprises 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.

32. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and the multispecific antibody of claim 29 and / or the immunoconjugate of claim 31.

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