Engineered antibodies and uses thereof

Abstract

Disclosed herein are isolated polypeptides and antibodies with improved sensitivity and reduced background signals and methods of using the same. In particular, these antibodies comprise a polypeptide comprising a CH2 domain defined by a sequence linked to a hinge domain.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of Indian Provisional Application No. IN202441066590, filed September 3, 2024; Indian Provisional Application No. IN202341061917, filed September 14, 2023; and Indian Provisional Application No. IN202341061835, filed September 14, 2023, under 35 USC § 119(e). The entire contents of the foregoing applications are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] This application contains a sequence list, which has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The copy of the .XML file, created on September 10, 2024, is named “TP385350WO1.xml” and has a size of 279,164 bytes. The sequence list contained in this .XML file is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0005] This invention relates to isolated peptides and antibodies with improved sensitivity and methods of using them. Background Technology

[0006] Recombinant antibodies offer the ability to be rationally engineered to produce beneficial properties. One such beneficial property is antibody sensitivity, i.e., the signal strength produced in a specific immunoassay. Highly sensitive antibodies have several advantages, such as the ability to detect low-abundance or rare targets and the ability to detect target proteins in challenging imaging assays. Antibody sensitivity can be increased through affinity maturation of the antibody clone, which involves mutations at the antigen-binding site or complementary site. However, affinity maturation must be tailored to each antibody in question and is therefore not a scalable solution for large numbers of antibodies. Furthermore, introducing mutations into the complementary site alters the antibody clone and essentially produces entirely new antibodies. Improved methods are needed to increase antibody sensitivity, such as through rational modification of the antibody's Fc region. The disclosed peptides, antibodies, compositions, and their uses address these and other important needs. Summary of the Invention

[0007] This article discloses isolated polypeptides containing the amino acid sequence of formula I: Z-(XY) n -Z r ) p-X, where Z is the hinge region, X is the CH2 domain, Y is the linker, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1. In some embodiments, the isolated peptide has at least a 0.5-fold increase in signal enhancement or sensitivity. In some embodiments, the isolated peptide has at least a 1 / 3 reduction in background signal.

[0008] This article discloses an antibody comprising a polypeptide linked to a Fab domain, the polypeptide comprising the amino acid sequence of formula I: Z-(XY) n -Z r ) p -X, where Z is the hinge region, X is the CH2 domain, Y is the linker, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1. In some embodiments, these antibodies have at least a 0.5-fold increase in signal enhancement or sensitivity compared to the corresponding wild-type antibody. In some embodiments, these antibodies have at least a 1 / 3 reduction in background signal compared to the corresponding wild-type antibody.

[0009] This article discloses antibodies that specifically bind to Parkin protein, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 13, an LCDR2 comprising the amino acid sequence RAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0010] This document discloses antibodies that specifically bind to OCT4, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 39; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0011] This document discloses antibodies that specifically bind to IDO1, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 66, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 68; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0012] This document discloses antibodies that specifically bind to FLAG tags, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 163, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 165; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 168, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 169; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0013] This article discloses rabbit anti-mouse (RAM) secondary antibodies, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 133; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge; and wherein the heavy chain further comprises a CH3 domain.

[0014] This document discloses antibodies that specifically bind to pERK1 / 2, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 91, an LCDR2 comprising the amino acid sequence of KVS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0015] This document discloses antibodies that specifically bind to PSD-95, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 111, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 113; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence of DTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 117; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and wherein the heavy chain further comprises a CH3 domain.

[0016] This document discloses antibodies that specifically bind to TIGIT, wherein these antibodies comprise a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 148; a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 151, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 152, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 152; wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge; and wherein the heavy chain further comprises a CH3 domain.

[0017] This document discloses a method for detecting the presence of a target protein in a sample, the method comprising applying an isolated peptide or antibody disclosed herein to a sample containing the target protein, incubating the isolated peptide or antibody in the sample under conditions that allow the isolated peptide or peptide or antibody to bind to the target protein and form a bound isolated peptide or bound peptide or antibody, and determining the presence of the bound isolated peptide or bound peptide or antibody, thereby detecting the presence of the target protein.

[0018] This article discloses methods for increasing the sensitivity of antibodies, which include introducing a polypeptide containing an amino acid sequence of formula I into the heavy chain of the antibody: Z-(XY) n -Z r ) p -X, where Z is a hinge, X is a CH2 domain, Y is a linker, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1, wherein the antibody's sensitivity is increased by at least 0.5-fold compared to the corresponding wild-type antibody. In some embodiments, the method further includes introducing an LFLEPS mutation into the amino acid sequence of X, wherein the LFLEPS mutation includes L234F, L235E, and P331S substitutions.

[0019] This article discloses methods for reducing background signal of antibodies, which include introducing a polypeptide containing an amino acid sequence of formula I into the heavy chain of the antibody: Z-(XY) n -Z r ) p-X, where Z is a hinge, X is a CH2 domain, Y is a linker, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1, wherein the background signal of the antibody is reduced by at least 1 / 3 compared to the corresponding wild-type antibody. In some embodiments, the method further includes introducing an LFLEPS mutation into the amino acid sequence of X, wherein the LFLEPS mutation includes L234F, L235E, and P331S substitutions. Attached Figure Description

[0020] The above overview and the following detailed description of preferred embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the precise embodiments shown in the figures.

[0021] Figure 1A This is a schematic diagram of the parental wild-type antibody (WT IgG, approximately 150 kDa).

[0022] Figure 1B This is a schematic diagram of a 1TdFc design (IgG-2Fc, approximately 200 kDa). The 1TdFc design features an additional Fc domain connected via a connector and hinge.

[0023] Figure 1C This is a schematic diagram of a 2TdFc design (IgG-3Fc, approximately 250 kDa). The 2TdFc design has two additional Fc domains connected by a connector and a hinge.

[0024] Figure 1D This is a schematic diagram of a 2TdCH2 design (approximately 200 kDa). The 2TdCH2 design features two additional CH2 structural domains connected via a joint and optional hinge.

[0025] Figure 1E This is a schematic diagram of the 2TdCH2-LFLEPS design (approximately 200 kDa). The 2TdCH2-LFLEPS design features two additional CH2 domains connected by a joint and optional hinge, as well as an FcR mutation (star-shaped) represented by the acronym "LFLEPS".

[0026] Figure 2A This is a plasmid map showing that a polynucleotide sequence of the OCT4 heavy chain (HC) with the 2TdCH2-LFLEPS conformation was synthesized and cloned between XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-KasI restriction enzyme can be used for subcloning of another target HC "Fab" region.

[0027] Figure 2BThis is a plasmid map showing that the polynucleotide sequence of the anti-OCT4 antibody light chain (LC) was synthesized and cloned between XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-NotI restriction enzyme can be used for subcloning of another target LC "Fab" region.

[0028] Figure 2C Restriction digestion analyses of anti-OCT4 antibody HC and anti-OCT4 antibody LC with the 2TdCH2-LFLEPS conformation are shown. Anti-OCT4 antibody HC 2TdCH2-LFLEPS showed the release of a 2203 bp insert fragment after XbaI-NotI digestion. Anti-OCT4 antibody LC showed the release of a 746 bp insert fragment after XbaI-NotI digestion.

[0029] Figure 3A This is a plasmid map showing that the polynucleotide sequence of the anti-Parkinsonin antibody heavy chain (HC) with the 2TdCH2-LFLEPS conformation was synthesized and cloned between the XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-KasI restriction enzyme can be used for subcloning of another target HC "Fab" region.

[0030] Figure 3B This is a plasmid map showing that the polynucleotide sequence of the anti-Parkinsonin antibody light chain (LC) was synthesized and cloned between the XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-NotI restriction enzyme can be used for subcloning of another target LC "Fab" region.

[0031] Figure 3C Restriction digestion analyses of anti-Parkinsonin antibody HC and anti-Parkinsonin antibody LC with the 2TdCH2-LFLEPS conformation are shown. Anti-Parkinsonin antibody HC 2TdCH2-LFLEPS showed the release of a 2218 bp insert fragment after XbaI-NotI digestion. Anti-Parkinsonin antibody LC showed the release of a 743 bp insert fragment after XbaI-NotI digestion.

[0032] Figure 4A Representative HPLC curves of parental anti-perkinin wild-type (WT) antibodies with 95% monomer purity are shown.

[0033] Figure 4B A representative HPLC curve of the anti-OCT4 1TdFc antibody with 63% monomer purity is shown.

[0034] Figure 4C A representative HPLC curve of the anti-Parkinsonin 2TdFc antibody with a monomer purity of 33% is shown.

[0035] Figure 4DA representative HPLC curve of the anti-Parkinsonin 2TdCH2-LFLEPS antibody with 87% monomer purity is shown.

[0036] Figure 5A Western blot (WB) analysis was performed on whole-cell extracts of rat brain tissue (left image) and whole-cell extracts of PC-12 cells (right image). Blots were detected using wild-type (WT) and engineered anti-Parkinsonin antibodies (1TdFC, 2TdFc, 2TdCH2). A band of approximately 48 kDa corresponding to Parkinsonin was observed in the tested rat brain and PC-12 cell lysates.

[0037] Figure 5B The image above shows a Western blot demonstrating antibody specificity through siRNA-mediated target protein knockdown. SH-SY5Y cells were transfected with Pakinin siRNA, and a decrease in signal intensity was observed in a Western blot application using an anti-Pakinin 2TdCH2-LFLEPS antibody. The image below is a graphical representation of the Western blot analysis.

[0038] Figure 5C Western blot analysis was performed on whole-cell extracts from PC-12 cells. Blots were detected using wild-type (WT) and engineered anti-Parkinsonin antibodies (2TdCH2, 2TdCH2-LFLEPS). Similar intensities of approximately 48 kDa bands corresponding to Parkinsonin were observed in PC-12 cell lysates using 2TdCH2 and 2TdCH2-LFLEPS.

[0039] Figure 5D Scatter plot analysis of WB data: Compared with WT antibody, anti-Parkinsonin 2TdCH2 antibody showed a significant fold increase (**p<0.01).

[0040] Figure 6A Western blot analysis was performed on whole-cell extracts from NTERA-2 cells (left) and F9 cells (right). The blots were detected using wild-type (WT) and engineered anti-OCT4 antibodies (1TdFC, 2TdFc, 2TdCH2). A band of approximately 38 kDa corresponding to OCT4 was observed in the cell lysates tested.

[0041] Figure 6B Western blot analysis was performed on whole-cell extracts from NTERA-2 cells. Blots were detected using wild-type (WT) and engineered anti-OCT4 antibodies (2TdCH2, 2TdCH2-LFLEPS). Similar intensities of approximately 38 kDa bands corresponding to OCT4 were observed in NTERA-2 cell lysates using both anti-OCT4 2TdCH2 and 2TdCH2-LFLEPS antibodies.

[0042] Figure 6C The antibody specificity is demonstrated by the relative expression of BOI in NTERA-2, NCCIT, and F9 cells (top panel). HEK293, A431, and HELA are negative cell lines. siRNA-mediated knockdown of the target protein is shown (bottom panel). NTERA-2 cells were transfected with OCT4 siRNA, and a decrease in signal intensity was observed in a Western blot application using the anti-OCT4 2TdCH2-LFLEPS antibody.

[0043] Figure 6D Scatter plot analysis of WB data: Compared with WT antibody, anti-OCT4 2TdCH2 antibody showed a significant fold increase (****p<0.0001).

[0044] Figure 7A This is a bar graph analysis of Western blot data obtained from the anti-Parkinsonin 2TdCH2 antibody and the various secondary antibodies shown. Compared with the wild-type (WT) antibody, the anti-Parkinsonin 2TdCH2 antibody showed a fold increase in different secondary antibodies.

[0045] Figure 7B This is a bar graph analysis of Western blot data obtained from the anti-OCT4 2TdCH2 antibody and the various secondary antibodies shown. Compared with the wild-type (WT) antibody, the anti-OCT4 2TdCH2 antibody showed a fold increase in protein blot data among the different secondary antibodies.

[0046] Figure 8A This is an immunofluorescence analysis of OCT4 performed using 70% confluence NTERA-2 cells. Cells were stained with different concentrations of anti-OCT4 wild-type (WT) antibody (top image) or anti-OCT4 2TdCH2-LFLEPS antibody (1:1,000) (bottom image).

[0047] Figure 8B The results showed that the siRNA (Silencer) was specifically validated. ® Product #s10871 and #s10873 were selected and transfected into NTERA-2 cells to achieve OCT4 knockdown. Immunofluorescence analysis was performed on NTERA-2 cells (untransfected, Fig. a-d), NTERA-2 cells transfected with OCT4-specific siRNA (Fig. i-l), or NTERA-2 cells transfected with non-specific mixed siRNA (Fig. e-h).

[0048] Figure 9A Immunohistochemical analysis of Parkin was performed using formalin-fixed paraffin-embedded mouse kidney tissue sections. The sections were probed with either anti-Parkinson wild-type antibody (WT) (bottom image) or anti-Parkinson 2TdCH2-LFLEPS antibody (top image) (1:100 dilution).

[0049] Figure 9B Immunohistochemical analysis of Parkin was performed using formalin-fixed paraffin-embedded mouse kidney tissue sections. The sections were probed with either an anti-Parkinson polyclonal antibody (benchmark (BM) or positive control) (bottom panel) or an anti-Parkinson 2TdCH2-LFLEPS antibody (top panel) (1:100 dilution).

[0050] Figure 10 Flow cytometry analysis of peradenin was performed in PC-12 cells. Cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer device (product number #88-8824-00), followed by intracellular staining with 2 μg of anti-peradenin wild-type (WT) antibody, anti-peradenin 2TdCH2-LFLEPS antibody, or positive control (PC) antibody (PA5-1339). Unstained samples served as controls. The top figure shows the flow cytometry spectra, and the bottom figure shows a graphical representation of the flow data.

[0051] Figure 11 This is a graphical representation of the immunofluorescence analysis of IDO1 performed on HEK-293E cells transfected with 70% confluent CD64-GFP (FcγR1), showing the amount of FcγR1 binding of the antibody. Cells were stained with 0.5 μg / ml and 2.5 μg / ml of IDO1 wild-type (WT) ± LFLEPS antibody or anti-IDO1 2TdCH2 ± LFLEPS antibody, followed by goat anti-rabbit Alexa Fluor. ™ Staining with 647 secondary antibody (product #A21244).

[0052] Figure 12A This is a flow cytometry analysis of IDO1 performed on HEK-293E cells transfected with 70% confluent CD64-GFP (FcγR1). Cells were stained with 0.5 μg / ml (bottom panel) and 2.5 μg / ml (top panel) anti-IDO1 wild-type ± LFLEPS (WT) antibody or anti-IDO1 2TdCH2 ± LFLEPS antibody, followed by goat anti-rabbit Alexa Fluor. ™ Staining with 647 secondary antibody (product #A21244). Analysis was performed using live cells and Attune. ™ Data was acquired using an NxT flow cytometer (product # A29004). A leftward shift in the histogram indicates a reduction in background signal following the LFLEPS mutation.

[0053] Figure 12B yes Figure 12AThe graphical representation of the flow cytometry analysis shown illustrates the amount of FcγR1 binding of the antibody. An asterisk (*) indicates statistical significance as determined by a p < 0.05 using a two-tailed Student's T test.

[0054] Figure 13A This is a graphical representation of the immunofluorescence analysis of IDO1 in HEK-293E cells transfected with 70% confluent CD64-GFP (FcγR1) in the presence and absence of interferon-γ (INFγ) treatment. Cells were stained with 2.5 µg / ml anti-IDO1 wild-type (WT) antibody ± LFLEPS or anti-IDO1 2TdCH2 ± LFLEPS antibody, followed by goat anti-rabbit Alexa Fluor. ™ Staining with 647 secondary antibody (product #A21244).

[0055] Figure 13B This is a graphical representation of the immunofluorescence analysis of IDO1 in HEK-293E cells transfected with 70% confluent CD64-GFP (FcγR1) with and without INFγ treatment. Cells were stained with 0.5 μg / ml anti-IDO1 wild-type (WT) antibody ± LFLEPS or anti-IDO1 2TdCH2 ± LFLEPS antibody, followed by goat anti-rabbit Alexa Fluor. ™ Staining with 647 secondary antibody (product #A21244).

[0056] Figure 14A This is a flow cytometry analysis demonstrating that the 2TdCH2-LFLEPS design in the mouse-rabbit chimeric antibody (Fab-Fc chimera) enhances the detection of TIGIT signal in human PBMCs compared to the wild-type design. Human PBMCs were stained with CD4 FITC (product #11-0049-42) and 0.25 μg of anti-TIGIT wild-type antibody, or anti-TIGIT+LFLEPS antibody, or anti-TIGIT+2TdCH2-LFLEPS antibody, followed by staining with goat anti-rabbit IgG (H+L) Alexa Fluor. ™ Staining with Plus 647 secondary antibody (product # A55060). Live cells from the lymphocyte phylum were used for analysis and Attune was applied. ™ Data was acquired using the NxT flow cytometer (product # A29004).

[0057] Figure 14B yes Figure 14AThe graphical representation of the flow cytometry analysis shown illustrates the staining indices of various antibodies. An asterisk (*) indicates statistical significance as determined by a two-tailed Student's t-test with p < 0.05, and an asterisk (**) indicates statistical significance as determined by a two-tailed Student's t-test with p < 0.01.

[0058] Figure 15 This is a graphical representation of the effect of 2TdCH2 LFLEPS design on rabbit anti-mouse (RAM) IgG secondary antibody using immunofluorescence analysis, showing the staining coefficients of various antibodies. Analysis was performed using HEK-293E cells transfected with 70% confluent CD64-GFP (FcγR1). Cells were stained with 0.5 μg / ml and 2.5 μg / ml RAM wild-type (WT) antibody ± LFLEPS or RAM2TdCH2-LFLEPS ± LFLEPS antibody, followed by donkey anti-rabbit Alexa Fluor. ™ 647 secondary antibody d (A32795) staining was used to detect RAM antibodies. Staining with RAM WT antibody and RAM 2TdCH2 antibody (without LFLEPS) increased the indication of nonspecific binding of the secondary antibody, since the primary antibody was not present in the sample. An asterisk (*) indicates statistical significance as determined by a p < 0.05 using a two-tailed Student's t-test.

[0059] Figure 16A The demonstration showed the use of 0.5 μg / ml anti-pERK1 / 2 WT antibody and anti-pERK1 / 2 2TdCH2 recombinant mouse monoclonal antibody, followed by goat anti-mouse IgG (H+L) horseradish peroxidase (HRP) superclonal antibody. ™ Western blot analysis of whole-cell extracts from Jurkat cells with and without PMA treatment (200 ng / ml) using secondary antibody (product #A28177) (left panel). The right panel shows a graphical representation of the Western blot analysis.

[0060] Figure 16B The treatment involved serum-starved A431 cells using 2.5 μg / ml anti-pERK1 / 2 WT antibody and anti-pERK1 / 22TdCH2 recombinant mouse monoclonal antibody, followed by goat anti-mouse IgG (H+L) Alexa Fluor. ™ Immunofluorescence analysis of the Plus 488 secondary antibody in the presence of EGF (200 ng / ml). The left image shows the immunofluorescence image and the right image shows a graphical representation of the immunofluorescence analysis.

[0061] Figure 17The use of anti-PSD-95 WT antibody and anti-PSD-95 2TdCH2 antibody at concentrations ranging from 4.0 μg / ml to 0.5 μg / ml was shown, followed by the use of goat anti-mouse IgG (H+L) HRP superclonal. ™ Secondary antibody (product # A28177) is a protein blot obtained by loading whole-cell extracts from Neuro2A cells.

[0062] Figure 18A In NTERA2 cells, 1 μg / ml wild-type or 2TdCH2 LFLEPS anti-OCT4-Alexa Fluor was used. ™ Immunofluorescence analysis of the Plus 555 antibody conjugate.

[0063] Figure 18B yes Figure 18A The graph shows a representation of the immunofluorescence analysis. An asterisk (**) indicates statistical significance as determined by a p < 0.01, as determined using a two-tailed Student's t-test.

[0064] Figure 19A In NTERA2 cells, 1 μg / ml wild-type or 2TdCH2 LFLEPS anti-OCT4-Alexa Fluor was used. ™ Immunofluorescence analysis of the Plus 647 conjugate.

[0065] Figure 19B yes Figure 19A The graph shows a representation of the immunofluorescence analysis. An asterisk (*) indicates statistical significance as determined by a p < 0.05, if a two-tailed Student's t-test is used.

[0066] Figure 20A Western blot analysis was performed on whole-cell lysates (WCL) of PC-12 cells using 1 μg / ml of wild-type or 2TdCH2LFLEPS anti-Parkinsonin HRP conjugate at molar ratios of 4 and 8.

[0067] Figure 20B Western blot analysis was performed in EXPI293 cells transfected with the FLAG-histone H4 construct using 1 μg / ml of wild-type or 2TdCH2 LFLEPS anti-FLAG tag HRP conjugate at molar ratios of 4 and 8.

[0068] Figure 20C Western blot analysis was performed in EXPI293 cells transfected with the FLAG-histone H4 construct using 1 μg / ml of wild-type or 2TdCH2 LFLEPS anti-FLAG-tagged biotin conjugate at molar ratios of 25 and 50.

[0069] Figure 21A Instructions from Alexa Fluor ™ SiteClick 488 dye with anti-perkinin WT, 2TdCH2 and 2TdCH2 LFLEPS antibodies ™ The degree of significance of the conjugation (DOL). An asterisk (*) indicates statistical significance as determined by a two-tailed Student's t-test with p < 0.05 and an asterisk (**) indicates statistical significance as determined by a two-tailed Student's t-test with p < 0.01.

[0070] Figure 21B Instructions from Alexa Fluor ™ SiteClick with 488 dye and anti-OCT4 WT, 2TdCH2 and 2TdCH2 LFLEPS antibodies ™ Dosage of the ligation (DOL). An asterisk (*) indicates statistical significance as determined by a p < 0.05 using a two-tailed Student's t-test. Detailed Implementation

[0071] Various publications, articles, and patents are cited or described in the background art and throughout the specification; these references are each incorporated herein by reference in their entirety. Discussions of documents, laws, materials, devices, articles, etc., already included in this specification are for the purpose of providing background to the invention. Such discussions are not an admission that any or all of these matters constitute prior art with respect to any disclosed or claimed invention.

[0072] The disclosed peptides, antibodies, and methods can be more readily understood by referring to the following detailed descriptions in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the disclosed peptides, antibodies, and methods are not limited to the specific peptides, antibodies, and methods described and / or illustrated herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed peptides, antibodies, and methods.

[0073] Unless otherwise specified, any description of possible mechanisms of action or modes of action or reasons for improvement is intended for illustrative purposes only, and the disclosed peptides, antibodies, and methods are not subject to the correctness or incorrectness of any such proposed mechanisms of action or modes of action or reasons for improvement.

[0074] Throughout this text, descriptions relate to peptides, antibodies, and methods for generating such peptides and antibodies. When this disclosure describes or claims features or embodiments related to peptides or antibodies, such features or embodiments also apply to methods for generating such peptides or antibodies. Similarly, when this disclosure describes or claims features or embodiments related to methods for generating peptides or antibodies, such features or embodiments also apply to such peptides or antibodies.

[0075] It should be understood that, for clarity, certain features of the disclosed peptides, antibodies, and methods described herein in the context of individual embodiments may also be provided in combination within a single embodiment. Conversely, for brevity, various features of the disclosed peptides, antibodies, and methods described in the context of a single embodiment may also be provided individually or in any sub-combination.

[0076] As used herein, the singular form “a” and “the” include the plural form.

[0077] When a list is presented, unless otherwise stated, it should be understood that each individual element of the list and each combination of the list is a separate implementation. For example, a list of implementations presented as “A, B or C” will be interpreted as including implementations: “A”; “B”; “C”; “A or B”; “A or C”; “B or C”; or “A, B or C”.

[0078] Various terms used throughout the specification and claims relate to the aspects described. Unless otherwise stated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0079] The term “comprising” is intended to include examples covered by the terms “substantially consisting of” and “consisting of”; similarly, the term “substantially consisting of” is intended to include examples covered by the term “consisting of”.

[0080] certain terms

[0081] As used herein, “antibody” means any IgG, IgM, IgA, IgD or IgE antibody or fragments or derivatives thereof derived from any animal, including but not limited to sheep, rabbits, goats, mice, camels and egg yolks.

[0082] Antibodies include, but are not limited to, monoclonal or polyclonal antibodies, bispecific antibodies, bifunctional antibodies, antibody fragments, humanized antibodies, chimeric antibodies, engineered antibodies, antibody constructs, and modified antibody-like structures. Engineered antibodies or antibody constructs include antibody-like structures having the polypeptides described herein, which comprise the amino acid sequence of Formula I: Z-(XY) n -Z r ) p-X, where Z is a hinge containing an amino acid sequence having at least 90% identity with SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102, X is a CH2 domain containing an amino acid sequence having at least 90% identity with SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y is a linker, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1. Antibodies can be produced or modified by recombinant DNA or other synthetic techniques. Antibodies can be antibodies isolated from mammalian serum samples, affinity-purified antibodies, or any mixture thereof, which exhibit sufficient binding specificity to the desired epitope or sequence derived therefrom. Functional derivatives or "equivalents" of antibodies are also included, such as single-chain antibodies, CDR transplantation antibodies, etc. VHH antibodies, which can be monovalent or bivalent, are also included.

[0083] As used herein, “biotin” means biotin or any biotin derivative, including but not limited to substituted and unsubstituted biotin and its analogues and derivatives, as well as substituted and unsubstituted derivatives of hexanobiotin, biocytin, desulfobiotin, desulfobiocytin, iminobiotin and biotin sulfone.

[0084] As used herein, “coding sequence” or “coding polynucleotide” means an RNA or DNA molecule that contains a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of a subject or mammal administered the nucleic acid molecule.

[0085] In the context of two or more nucleic acid molecules or polypeptide sequences, as used herein, “identity” or “identity percentage” means that the sequences have a specified percentage of identical residues in a specified region. This percentage can be calculated by: optimally aligning the two sequences, comparing them in a specified region, determining the number of positions in both sequences where the same base occurs to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences have different lengths, or where the alignment produces one or more staggered ends, and the specified region being compared includes only a single sequence, the residues of that single sequence are included in the denominator rather than the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0086] As used herein, a "fluorophore" refers to a compound that, when excited by exposure to light of a specific wavelength, emits light (fluorescence) at a wavelength different from the wavelength to which it is exposed. Fluorophores may contain substituents that alter their solubility, spectral properties, or physical characteristics.

[0087] Exemplary fluorophores include, but are not limited to, xanthocyanin, fluorescein, rhodamine, silanol, rhodol, roseamine, carbopyranone, indole, indacene, boron polyacrylamide, furan, benzofuran, anthocyanin, benzopyrilium, pyrene, coumarin, styryl, squarine, resorufin, anthraquinone, acridine, benzophenoxazine, quinoline, quinazolinone, oligonucleotide-based fluorescent dyes, conjugated polymer dyes, and tandem dyes.

[0088] Examples of fluorophores suitable for use with the disclosed assays include, but are not limited to, 6-carboxyfluorescein (FAM), tetrachlorofluorescein (TET), tetramethylrhodamine (TMR), hexachlorofluorescein (HEX), JOE, 6-carboxy-X-rhodamine (ROX), and CAL Fluor. ™ Pulsar ™ Quasar ™ Texas Red ™ Texas Red-X, Cy ™ 3. Cy ™ 5. BODIPY ™ (Boron-methylene dipyrrole), Alexa Fluor ™ Dyes (such as Alexa Fluor) ™ 488), Alexa Fluor Plus dyes (such as Alexa Fluor) ™ Plus 488), DyLight ™ Dyes (such as DyLight) ™ 488), Oregon Green ™ and NovaFluor ™ Dyes (such as NovaFluor) ™Red 710), and other fluorophores described in the following literature: Johnson, I. and Spence, MTZ (ed.) (2010) The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th edition.

[0089] As used herein, “immunospecific binding” refers to the ability of a disclosed peptide or antibody to preferentially bind to its target rather than to other molecules in a sample or environment containing a mixed molecular population.

[0090] As used herein, “label” means a reagent that can be detected, for example, by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached (e.g., covalently linked or conjugated) to a nucleic acid molecule or protein (such as an antibody or its antigen-binding fragment), thereby allowing the detection of that nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Specific, non-limiting examples of labels include fluorescent portions and fluorophore portions (e.g., fluorophores).

[0091] Illustrative markings include markings that can be directly observed or measured, or indirectly observed or measured. Such markings include, but are not limited to, radioactive markings that can be measured using a radiation counting device; pigments, dyes, or other chromogens that can be visually observed or measured using a spectrophotometer; spin markings that can be measured using a spin marking analyzer; and fluorescent portions, wherein the output signal is generated by exciting a suitable molecular adduct and can be visualized by excitation with light absorbed by a dye or measured using, for example, a standard fluorometer or imaging system.

[0092] The label can be a luminescent substance, such as a phosphor or fluorophore; a bioluminescent substance; a chemiluminescent substance, wherein the output signal is generated by chemical modification of a signaling compound; a metal-containing substance; or an enzyme, wherein an enzyme-dependent secondary signal is generated, such as the formation of a colored product from a colorless substrate. The label can also take the form of chemical or biochemical or inert particles, including but not limited to colloidal gold, microspheres, quantum dots, or inorganic crystals such as nanocrystals or phosphors (see, for example, Beverloo et al. Anal. Biochem. 203, 326-34 (1992)).

[0093] The term "label" can also refer to a "tag" or hapten that can selectively bind to a labeled molecule, allowing the labeled molecule to generate a detectable signal upon subsequent addition. For example, biotin, iminobiotin, or dethiobiotin can be used as a tag, which is then bound to a horseradish peroxidase (HRP) avidin or a streptomycin conjugate, followed by a chromogenic substrate (e.g., tetramethylbenzidine) or a fluorescent substrate such as Amplex. ™ Red or Amplex ™ Gold (Thermo Fisher Scientific, Waltham, MA) detects the presence of HRP. Similarly, the tag can be a hapten or antigen (e.g., isohydroxydigitoxin), and enzyme-catalyzed, fluorescently, or radiolabeled antibodies can be used to bind to the tag.

[0094] Many markers are known to those skilled in the art, including but not limited to particles, fluorescent dyes, haptens, enzymes and their chromogenic, fluorescent and chemiluminescent substrates, as well as other markers described in the following literature: Johnson, I. and Spence, MTZ (ed.) (2010) The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th edition.

[0095] As used herein, “nucleic acid molecule,” “oligonucleotide,” or “polynucleotide” refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, a nucleic acid molecule also encompasses the complementary strand of the single strand described. Many variants of a nucleic acid molecule can be used for the same purpose as a given nucleic acid molecule. Therefore, a nucleic acid molecule also encompasses substantially the same nucleic acid molecule and its complement.

[0096] Nucleic acid molecules can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acid molecules can be DNA (genomic and cDNA), RNA, or hybrids. They can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acid molecules can undergo post-transcriptional chemical modifications. They can be obtained through chemical synthesis or recombination.

[0097] As used herein, "oligonucleotide-based fluorescent dyes" refers to nucleic acid nanostructures constructed from DNA-based scaffolds, which precisely arrange fluorophores to engineer their interactions and the overall fluorescence properties of the structure. Examples of oligonucleotide-based fluorescent dyes are described in International Patent Application Publication No. WO 2018 / 231805, and include, but are not limited to, NovaFluor. ™ Dyes (Thermo Fisher Scientific, Waltham, MA).

[0098] As used herein, “peptide,” “protein,” or “polypeptide” can refer to a sequence of linked amino acids and can be natural, synthetic, or a combination of natural and synthetic modifications. Peptides, proteins, or polypeptides can have post-translational modifications.

[0099] As used herein, "vector" refers to a polynucleotide sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. A vector may contain or include one or more heterologous polynucleotide sequences.

[0100] engineered antibodies

[0101] Recombinant antibodies offer the ability to engineer benefits in a rational manner. One of the most important characteristics of an antibody is its sensitivity, i.e., the signal strength produced in a specific immunoassay. Highly sensitive antibodies have several benefits, such as the detection of low-abundance or rare targets and the ability to detect proteins of interest in complex assays such as imaging. Antibody sensitivity is affected by both increased signal and reduced background. Antibody sensitivity can be increased through affinity maturation of the antibody clone, which involves mutations at the antigen-binding site or complementary site. However, affinity maturation must be tailored to each antibody and is therefore not a scalable solution for large numbers of antibodies. Furthermore, mutations at the complementary site alter the antibody clone and essentially create entirely new antibodies.

[0102] This disclosure provides a method for increasing antibody sensitivity through rational modification of the Fc region of an antibody. Amplification or extension of the entire Fc domain or a portion thereof provides additional binding sites for secondary antibodies, resulting in enhanced signal. Previous studies have used Fc amplification for other purposes, such as, in the case of humanized antibodies, to increase antibody efficacy in therapeutic applications by enhancing binding to the Fc receptor. Antibody modifications by adding one additional Fc domain (1TdFc) or two additional Fc domains (2TdFc) to the antibody have been found to result in poor expression, leading to low yields and challenges in aggregation and purification. To overcome these limitations, this disclosure provides a rational design to circumvent the drawbacks of previous studies by selectively adding an additional CH2 domain (2TdCH2) instead of the entire Fc region. The 2TdCH2 design of the peptides and antibodies of the present invention provided herein overcomes these challenges, and it has been unexpectedly found that the 2TdCH2 engineered antibodies provided herein exhibit yields, monomeric purity, and stability comparable to wild-type antibodies. Two additional CH2 domains, connected by hinges and linkers, provide additional epitopes for secondary antibody binding and additional lysine residues for conjugation. Furthermore, the 2TdCH2 engineered antibodies of this disclosure offer a significant increase in sensitivity in a variety of applications, such as Western blotting and immunocytochemistry, and allow for a wider range of antibody applicability. In addition to the benefits of unconjugated antibodies, 2TdCH2 engineering provides additional binding sites for different conjugation chemistry. It was also unexpectedly found that introducing specific mutations L234F, L235E, and P331S (described herein as the “LFLEPS mutation”) into the engineered backbone reduced background binding of the antibody to the Fcγ receptor present on selected cells. Therefore, the advantages of the engineered antibodies of this disclosure include: i) an increase in sensitivity of at least 0.5-fold; ii) a background reduction of up to 1 / 3; iii) detection of low-abundance targets that are difficult to detect with standard antibodies; iv) increased application coverage through the use of multiplexing; iv) increased conjugation sites for primary conjugates; and v) resolution of design challenges related to expression, purity, and stability found with other recombinant antibodies.

[0103] This article discloses isolated polypeptides comprising an amino acid sequence of Formula I, substantially composed of an amino acid sequence of Formula I, or composed of an amino acid sequence of Formula I: Z-(XY) n -Z r ) p -X, where Z is the hinge region, X is the CH2 structural domain, Y is the joint, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1. In some implementations, this is defined as Z-(XY) n -Z r ) p The amino acid sequence elements of Formula I of -X are directly linked to each other, without any inserted amino acid sequences.

[0104] The hinge (Z) region may contain the amino acid sequence of SEQ ID NO: 18. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. The hinge (Z) may contain the amino acid sequence of SEQ ID NO: 48. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. The hinge (Z) may contain the amino acid sequence of SEQ ID NO: 102. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0105] The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 19. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 49. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 104. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0106] The isolated polypeptide may contain one or more adapters (Y) comprising the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The one or more adapters (Y) may contain GS. The one or more adapters (Y) may contain GGS. The one or more adapters (Y) may contain SEQ ID NO: 14. The one or more adapters (Y) may contain SEQ ID NO: 38. The one or more adapters (Y) may contain SEQ ID NO: 43. The one or more adapters (Y) may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The one or more adapters (Y) may contain SEQ ID NO: 44. The one or more adapters (Y) may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0107] The isolated polypeptide can contain one linker (Y) such that n is 1. The isolated polypeptide can contain two linkers (Y) such that n is 2. The isolated polypeptide can contain three linkers (Y) such that n is 3. The isolated polypeptide can contain four linkers (Y) such that n is 4. The isolated polypeptide can contain five linkers (Y) such that n is 5.

[0108] The isolated polypeptide may contain two or more CH2-linker regions ((XY)). n ) p The region contains two CH2-linker regions ((XY)) such that p is 2 or greater. The isolated polypeptide may contain two CH2-linker regions ((XY)). n ) p ), making p = 2. The isolated polypeptide can contain three CH2-linker regions ((XY) n ) p ), making p = 3. The isolated polypeptide can contain four CH2-linker regions ((XY) n ) p ), making p = 4. The isolated polypeptide can contain five CH2-linker regions ((XY) n ) p ), making p = 5.

[0109] The isolated polypeptide may contain two or more CH2-linker-hinge regions ((XY) n -Z r ) p The region contains two CH2-linker-hinge regions ((XY)) such that p is 2 or greater. The isolated polypeptide may contain two CH2-linker-hinge regions ((XY)). n -Z r ) p), making p = 2. The isolated polypeptide can contain three CH2-linker-hinge regions ((XY)). n -Z r ) p ), making p = 3. The isolated polypeptide can contain four CH2-linker-hinge regions ((XY) n -Z r ) p ), making p = 4. The isolated polypeptide can contain five CH2-linker-hinge regions ((XY) n -Z r ) p ), making p = 5.

[0110] In some implementations, the isolated polypeptide comprises (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 18, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 19, Y (linker) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0111] In some implementations, the isolated polypeptide comprises (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 48, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 49, Y (connector) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0112] In some implementations, the isolated polypeptide comprises (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 104, Y (connector) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0113] In some implementations, the isolated polypeptide comprises (XY) n -Z r ) pIt is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 104, Y (linker) contains the amino acid sequence of SEQ ID NO: 45, wherein n is 1, r is 0 or 1, and p is 2.

[0114] In some embodiments, the isolated polypeptide comprises two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may comprise the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated polypeptide comprising a CH3 domain comprises SEQ ID NO: 52. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 52. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 52.

[0115] In some embodiments, the isolated polypeptide comprises two additional CH2 structures at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and includes an “LFLEPS” mutation within the lower hinge and CH2 domain. The “LFLEPS” mutation may comprise the following amino acid substitutions: L234F, L235E, and P331S. An isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and including an “LFLEPS” mutation within the lower hinge and CH2 domain, comprises SEQ ID NO: 54. An isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and including an “LFLEPS” mutation within the lower hinge and CH2 domain, may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 54. An isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain connected via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domain, may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 54.

[0116] Any isolated polypeptide disclosed herein may be linked to a second polypeptide via one or more disulfide bonds, wherein the isolated polypeptide and the second polypeptide share the same amino acid sequence. These one or more disulfide bonds may be present between the hinges of the isolated polypeptide and the hinges of the second polypeptide.

[0117] Any isolated polypeptide disclosed herein may also contain a CH3 domain. In some embodiments, the isolated polypeptide disclosed herein may also contain no more than one CH3 domain. In some embodiments, the CH3 domain contains the amino acid sequence of SEQ ID NO: 20. The CH3 domain may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The CH3 domain may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0118] In some embodiments, the isolated polypeptide containing the CH3 domain comprises SEQ ID NO: 106. The isolated polypeptide containing the CH3 domain may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 106. The isolated polypeptide containing the CH3 domain may comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 106.

[0119] In some embodiments, the isolated polypeptide containing the CH3 domain comprises SEQ ID NO: 141. The isolated polypeptide containing the CH3 domain may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 141. The isolated polypeptide containing the CH3 domain may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 141.

[0120] This document also provides antibodies comprising any isolated peptide disclosed herein linked to a Fab domain. The Fab may comprise a CH1 domain. The CH1 domain may comprise the amino acid sequence of SEQ ID NO: 17. The CH1 domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17. The CH1 domain may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 17. The Fab may comprise a Fab heavy chain and a Fab light chain paired to form a binding domain.

[0121] In some embodiments, Fab may comprise a CH1 domain. The CH1 domain may comprise the amino acid sequence of SEQ ID NO: 100. The CH1 domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 100. The CH1 domain may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 100. Fab may comprise a Fab heavy chain and a Fab light chain paired to form a binding domain.

[0122] In some embodiments, Fab may comprise a CH1 domain. The CH1 domain may comprise the amino acid sequence of SEQ ID NO: 160. The CH1 domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 160. The CH1 domain may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 160. Fab may comprise a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0123] In some embodiments, antibodies comprising any isolated peptide disclosed herein linked to a Fab domain have a background signal reduction of at least 1 / 3 compared to the corresponding wild-type antibody. In some embodiments, the background signal reduction is at least 1 / 3, at least 1 / 2, at least 3 / 5, at least 2 / 3, at least 5 / 7, at least 3 / 4, at least 7 / 9, or at least 4 / 5 compared to the corresponding wild-type antibody.

[0124] In some embodiments, antibodies comprising any isolated peptide disclosed herein linked to a Fab domain exhibit at least a 0.5-fold increase in sensitivity compared to the corresponding wild-type antibody. In some embodiments, the increase in sensitivity is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, or at least 9-fold compared to the corresponding wild-type antibody.

[0125] In some embodiments, the antibody comprising any isolated peptide disclosed herein linked to the Fab domain has at least a 0.5-fold increase in signal enhancement compared to the corresponding wild-type antibody. In some embodiments, the increase in signal enhancement is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, or at least 9-fold compared to the corresponding wild-type antibody.

[0126] The Fab of an antibody can bind specifically to one or more target molecules. The Fab can bind specifically to perkinin, OCT4, IDO1, FLAG tag, pERK1 / 2, PSD-95, or TIGIT, or the Fab can be a rabbit anti-mouse (RAM) IgG secondary antibody.

[0127] In some embodiments, Fab binds specifically to the peracinin immune system. The Fab that binds specifically to the peracinin immune system may comprise a Fab heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments of this disclosure, the CDRs are defined according to the IMGT system.

[0128] The Fab that specifically binds to Parkin can comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 7. The Fab that specifically binds to Parkin can comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7. The Fab that specifically binds to Parkin can comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0129] The Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 9. The Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. The Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9.

[0130] The Fab that specifically binds to the perkinin may comprise a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 13, an LCDR2 containing the amino acid sequence RAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0131] The Fab that specifically binds to the peradenine may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 16. The Fab that specifically binds to the peradenine may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. The Fab that specifically binds to the peradenine may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0132] The Fab that specifically binds to the Parkinson protein may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 12. The Fab that specifically binds to the Parkinson protein may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12. The Fab that specifically binds to the Parkinson protein may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 12.

[0133] The Fab that specifically binds to the perkinin may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 4, an HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 6. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 13, an LCDR2 containing the amino acid sequence RAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0134] The Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 7, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 16. Alternatively, the Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16. Another option is to specifically bind to the Parkinson protein by comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0135] The Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 12. Alternatively, the Fab that specifically binds to the Parkinson protein may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12. Another option is to specifically bind to the Parkinson protein by comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12.

[0136] In some embodiments, the antibody comprising a Fab that specifically binds to the peradenine protein comprises the amino acid sequence of SEQ ID NO: 3. The antibody comprising a Fab that specifically binds to the peradenine protein may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3. The antibody comprising a Fab that specifically binds to the peradenine protein may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3.

[0137] In some embodiments, Fab binds specifically to OCT4 immune responses. Fab that binds specifically to OCT4 immune responses may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31.

[0138] The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 32. The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32. The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32.

[0139] The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO:34. The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:34. The Fab that specifically binds to OCT4 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:34.

[0140] The Fab that specifically binds to OCT4 may comprise a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO:37, an LCDR2 containing the amino acid sequence GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO:39.

[0141] The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 40. The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40.

[0142] The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO:36. The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:36. The Fab that specifically binds to OCT4 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:36.

[0143] The Fab that specifically binds to OCT4 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 29, an HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 31. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 37, an LCDR2 containing the amino acid sequence GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0144] The Fab that specifically binds to OCT4 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 32, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 40. Alternatively, the Fab that specifically binds to OCT4 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 40. Another option is to include a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0145] The Fab that specifically binds to OCT4 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 36. Alternatively, the Fab that specifically binds to OCT4 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 36. Another option is to make the Fab that specifically binds to OCT4 immunely, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 36.

[0146] In some embodiments, the antibody comprising a Fab that specifically binds to OCT4 immunity comprises the amino acid sequence of SEQ ID NO: 28. The isolated polypeptide comprising a Fab that specifically binds to OCT4 immunity may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28. The isolated polypeptide comprising a Fab that specifically binds to OCT4 immunity may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 28.

[0147] In some embodiments, Fab binds specifically to IDO1 immunely. Fab that binds specifically to IDO1 immunely may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 66, an HCDR2 containing the amino acid sequence of SEQ ID NO: 67, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 68.

[0148] The Fab that specifically binds to IDO1 may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 65. The Fab that specifically binds to IDO1 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65. The Fab that specifically binds to IDO1 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65.

[0149] The Fab that specifically binds to IDO1 may comprise a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 72, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 73.

[0150] The Fab that specifically binds to IDO1 may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 71. The Fab that specifically binds to IDO1 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. The Fab that specifically binds to IDO1 may comprise a Fab light chain containing a variable domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0151] The Fab that specifically binds to IDO1 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 66, an HCDR2 containing the amino acid sequence of SEQ ID NO: 67, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 68. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 72, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 73.

[0152] The Fab that specifically binds to IDO1 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 65, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 71. Alternatively, the Fab that specifically binds to IDO1 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71. Another option is to make the Fab that specifically binds to IDO1 immunely, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0153] In some embodiments, the antibody comprising a Fab that specifically binds to IDO1 comprises the amino acid sequence of SEQ ID NO: 79. The isolated polypeptide comprising a Fab that specifically binds to IDO1 may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 79. The isolated polypeptide comprising a Fab that specifically binds to IDO1 may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 79.

[0154] In some implementations, Fab binds immunospecifically to the FLAG tag. The Fab that binds immunospecifically to the FLAG tag may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 163, an HCDR2 containing the amino acid sequence of SEQ ID NO: 164, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 165.

[0155] The Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 162. The Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162. The Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162.

[0156] The Fab that binds specifically to the FLAG tag may comprise a Fab light chain containing: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 168, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 169.

[0157] An immune-specific FLAG-tag-binding Fab may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 167. An immune-specific FLAG-tag-binding Fab may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167. An immune-specific FLAG-tag-binding Fab may comprise a Fab light chain containing a variable domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167.

[0158] The Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 163, an HCDR2 containing the amino acid sequence of SEQ ID NO: 164, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 165. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 168, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 169.

[0159] The Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 162, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 167. Alternatively, the Fab that specifically binds to the FLAG tag may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 167. Another option is to make the Fab that specifically binds to the FLAG tag immunely by comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 167.

[0160] In some embodiments, the antibody comprising a Fab that specifically binds to a FLAG tag comprises the amino acid sequence of SEQ ID NO: 175. The isolated polypeptide comprising a Fab that specifically binds to a FLAG tag may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 175. The isolated polypeptide comprising a Fab that specifically binds to a FLAG tag may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 175.

[0161] In some embodiments, Fab is a rabbit anti-mouse IgG secondary antibody (also referred to herein as "rabbit anti-mouse IgG antibody", "rabbit anti-mouse IgG", or "RAM"). Fab as RAM may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129.

[0162] The Fab as RAM may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 126. The Fab as RAM may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126. The Fab as RAM may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126.

[0163] The Fab as RAM may contain a Fab light chain comprising: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, an LCDR2 containing the amino acid sequence of GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 133.

[0164] The Fab as RAM may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 131. The Fab as RAM may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 131. The Fab as RAM may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 131.

[0165] The Fab as RAM may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 127, an HCDR2 containing the amino acid sequence of SEQ ID NO: 128, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 129. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, an LCDR2 containing the amino acid sequence of GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 133.

[0166] The Fab as RAM may comprise a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 126, and the Fab light chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 131. Alternatively, the Fab as RAM may comprise a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126, and the Fab light chain comprises a variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 131. Finally, the Fab as RAM may comprise a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126, and the Fab light chain comprises a variable region comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 131.

[0167] In some embodiments, the antibody comprising the Fab as RAM comprises the amino acid sequence of SEQ ID NO: 139. The isolated polypeptide comprising the RAM may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 139. The isolated polypeptide comprising the RAM may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 139.

[0168] In some embodiments, Fab binds specifically to pERK1 / 2 immunely. Fab that binds specifically to pERK1 / 2 immunely may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0169] The Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 84. The Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84. The Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84.

[0170] The Fab that specifically binds to pERK1 / 2 may comprise a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, an LCDR2 containing the amino acid sequence of KVS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0171] The Fab that specifically binds to pERK1 / 2 may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 90. The Fab that specifically binds to pERK1 / 2 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. The Fab that specifically binds to pERK1 / 2 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0172] The Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 85, an HCDR2 containing the amino acid sequence of SEQ ID NO: 86, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 87. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, an LCDR2 containing the amino acid sequence of KVS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0173] The Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 84, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 90. Alternatively, the Fab that specifically binds to pERK1 / 2 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. Another option is to include a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0174] In some embodiments, the antibody comprising a Fab that specifically binds to pERK1 / 2 contains the amino acid sequence of SEQ ID NO:98. The isolated polypeptide comprising a Fab that specifically binds to pERK1 / 2 may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO:98. The isolated polypeptide comprising a Fab that specifically binds to pERK1 / 2 may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO:98.

[0175] In some embodiments, Fab binds specifically to PSD-95 immunely. Fab that binds specifically to PSD-95 immunely may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 111, an HCDR2 containing the amino acid sequence of SEQ ID NO: 112, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 113.

[0176] The Fab that specifically binds to PSD-95 may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 110. The Fab that specifically binds to PSD-95 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110. The Fab that specifically binds to PSD-95 may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110.

[0177] The Fab that specifically binds to PSD-95 may comprise a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116, an LCDR2 containing the amino acid sequence of DTS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 117.

[0178] The Fab that specifically binds to PSD-95 may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 115. The Fab that specifically binds to PSD-95 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. The Fab that specifically binds to PSD-95 may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115.

[0179] The Fab that specifically binds to PSD-95 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 111, an HCDR2 containing the amino acid sequence of SEQ ID NO: 112, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 113. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116, an LCDR2 containing the amino acid sequence of DTS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 117.

[0180] The Fab that specifically binds to PSD-95 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 110, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 115. Alternatively, the Fab that specifically binds to PSD-95 may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 115. Another option is to include a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 115.

[0181] In some embodiments, the antibody comprising a Fab that specifically binds to PSD-95 immunity comprises the amino acid sequence of SEQ ID NO: 123. The isolated polypeptide comprising a Fab that specifically binds to PSD-95 immunity may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 123. The isolated polypeptide comprising a Fab that specifically binds to PSD-95 immunity may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 123.

[0182] In some embodiments, Fab binds to TIGIT immune-specifically. Fab that binds to TIGIT immune-specifically may comprise a heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 148.

[0183] The Fab that specifically binds to TIGIT may comprise a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 145. The Fab that specifically binds to TIGIT may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145. The Fab that specifically binds to TIGIT may comprise a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145.

[0184] The Fab that specifically binds to TIGIT may comprise a Fab light chain containing: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151, an LCDR2 containing the amino acid sequence of SAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 152.

[0185] The Fab that specifically binds to TIGIT may comprise a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 150. The Fab that specifically binds to TIGIT may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. The Fab that specifically binds to TIGIT may comprise a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0186] The Fab that specifically binds to TIGIT may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 146, an HCDR2 containing the amino acid sequence of SEQ ID NO: 147, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 148. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151, an LCDR2 containing the amino acid sequence of SAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 152.

[0187] The Fab that specifically binds to TIGIT may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 145, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 150. Alternatively, the Fab that specifically binds to TIGIT may comprise a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. Another option is to include a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0188] In some embodiments, the Fab that specifically binds to TIGIT comprises the amino acid sequence of SEQ ID NO: 157. An isolated polypeptide comprising the Fab that specifically binds to TIGIT may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 157. An isolated polypeptide comprising the Fab that specifically binds to TIGIT may comprise an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 157.

[0189] Any antibody disclosed herein may be an antibody having the same type of IgG.

[0190] In some implementations, the isolated peptides or antibodies also contain a label.

[0191] This document discloses polynucleotides encoding isolated polypeptides, nucleic acid molecules substantially composed of or composed of such polynucleotides, wherein the isolated polypeptides contain, substantially consist of, or consist of an amino acid sequence of Formula I: Z-(XY) n -Z r ) p -X, where Z is the hinge, X is the CH2 structural domain, Y is the joint, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1.

[0192] The nucleic acid molecule may contain a polynucleotide encoding a hinge (Z) comprising the amino acid sequence of SEQ ID NO: 18. The polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the hinge (Z) comprises SEQ ID NO: 22.

[0193] Nucleic acid molecules can contain polynucleotides encoding polypeptides, which contain (XY) n -Z r ) p The device is substantially composed of or consists of the hinge (Z), wherein the hinge (Z) comprises the amino acid sequence of SEQ ID NO: 48. The polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, the polynucleotide encoding the hinge (Z) comprises SEQ ID NO: 51.

[0194] Nucleic acid molecules can contain polynucleotides encoding polypeptides, which contain (XY) n -Z r The polynucleotide may be substantially composed of or comprised of the same, wherein the hinge (Z) comprises the amino acid sequence of SEQ ID NO: 102. The polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. Alternatively, the polynucleotide may encode a hinge (Z) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102. In some embodiments, the polynucleotide encoding the hinge (Z) comprises SEQ ID NO: 101.

[0195] The encoding includes (XY) n -Z r ) pA polynucleotide comprising or consisting substantially of a polypeptide may encode a CH2 domain (X) containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide may encode a CH2 domain (X) containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide may encode a CH2 domain (X) containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 23 or SEQ ID NO: 178.

[0196] The encoding includes (XY) n -Z r ) p Nucleic acid molecules consisting substantially of or composed of polypeptides thereof may contain a polynucleotide encoding a CH2 domain (X) comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may encode a CH2 domain (X) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a CH2 domain (X) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 62.

[0197] The encoding includes (XY) n -Z r ) p A polynucleotide comprising or consisting substantially of a polypeptide may encode a CH2 domain (X) comprising the amino acid sequence of SEQ ID NO: 102. A polynucleotide may encode a CH2 domain (X) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. A polynucleotide may encode a CH2 domain (X) comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102. In some embodiments, the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 101.

[0198] The encoding includes (XY) n -Z r ) pA nucleic acid molecule consisting essentially of or composed of a polypeptide may contain a polynucleotide encoding an isolated polypeptide comprising one or more adapters (Y), wherein the one or more adapters comprises the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The polynucleotide may encode one or more adapters (Y) comprising GS. The polynucleotide may encode one or more adapters (Y) comprising GGS. The polynucleotide may encode one or more adapters (Y) comprising SEQ ID NO: 14. The polynucleotide may encode one or more adapters (Y) comprising SEQ ID NO: 38. The polynucleotide may encode one or more adapters (Y) comprising SEQ ID NO: 43. The polynucleotide may encode one or more adapters (Y) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 43. The polynucleotide may encode one or more adapters (Y) comprising SEQ ID NO: 44. The polynucleotide may encode one or more adapters (Y) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44. In some implementations, the polynucleotide encoding the one or more adapters (Y) comprises SEQ ID NO: 46.

[0199] The polynucleotide can encode a separate polypeptide containing one linker (Y), such that n is 1. The polynucleotide can encode a separate polypeptide containing two linkers (Y), such that n is 2. The polynucleotide can encode a separate polypeptide containing three linkers (Y), such that n is 3. The polynucleotide can encode a separate polypeptide containing four linkers (Y), such that n is 4. The polynucleotide can encode a separate polypeptide containing five linkers (Y), such that n is 5.

[0200] This polynucleotide can encode a region containing two or more CH2-linker regions ((XY)). n ) p A polypeptide isolated from the region of ), such that p is 2 or greater. This polynucleotide can encode a region containing two CH2-connector regions ((XY)). n ) p The isolated polypeptide has a p=2. This polynucleotide can encode a region containing three CH2-connector regions ((XY)). n ) p The isolated polypeptide has a p=3. This polynucleotide can encode a region containing four CH2-connector regions ((XY)). n ) p The isolated polypeptide has a p=4. This polynucleotide can encode a region containing five CH2-connector regions ((XY)). n )p The isolated polypeptides have a p=5.

[0201] This polynucleotide can encode two or more CH2-linker-hinge regions ((XY)). n -Z r ) p A polypeptide consisting of two CH2-linker-hinge regions ((XY)) such that p is 2 or greater. This polynucleotide can encode a region containing two CH2-linker-hinge regions ((XY)). n -Z r ) p The isolated polypeptide has a p=2. This polynucleotide can encode a region containing three CH2-linker-hinge regions ((XY)). n -Z r ) p The isolated polypeptide has a p=3. This polynucleotide can encode a region containing four CH2-linker-hinge regions ((XY)). n -Z r ) p The isolated polypeptide has a p=4. This polynucleotide can encode a region containing five CH2-linker-hinge regions ((XY)). n -Z r ) p The isolated polypeptides have a p=5.

[0202] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide comprising, substantially comprising, or comprising the following: Z (hinge) containing the amino acid sequence of SEQ ID NO: 18, X (CH2 domain) containing the amino acid sequence of SEQ ID NO: 19, and Y (linker) containing the amino acid sequence of SEQ ID NO: 44, wherein n is 1 and p is 2. In some embodiments, the polynucleotide encoding the hinge (X) comprises SEQ ID NO: 22 and the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 23 or SEQ ID NO: 178.

[0203] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide comprising, substantially comprising, or comprising the following: Z (hinge) containing the amino acid sequence of SEQ ID NO: 48, X (CH2 domain) containing the amino acid sequence of SEQ ID NO: 49, and Y (linker) containing the amino acid sequence of SEQ ID NO: 44, wherein n is 1 and p is 2. In some embodiments, the polynucleotide encoding the hinge (X) comprises SEQ ID NO: 51 and the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 62.

[0204] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide comprising, substantially comprising, or comprising the following: a Z (hinge) containing the amino acid sequence of SEQ ID NO: 102, an X (CH2 domain) containing the amino acid sequence of SEQ ID NO: 104, and a Y (linker) containing the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2. In some embodiments, the polynucleotide encoding the hinge (X) comprises SEQ ID NO: 101 and the polynucleotide encoding the CH2 domain (X) comprises SEQ ID NO: 103.

[0205] The polynucleotide can encode a polypeptide comprising two additional CH2 domains at the N-terminus of an Fc domain linked via a hinge and optionally one or more linkers, wherein the separated polypeptide comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the polynucleotide encodes a polypeptide comprising two additional CH2 domains at the N-terminus of an Fc domain linked via a hinge and optionally one or more linkers, wherein the separated polypeptide comprises SEQ ID NO: 52. The polynucleotide can encode a polypeptide comprising two additional CH2 domains at the N-terminus of an Fc domain linked via a hinge and optionally one or more linkers, wherein the separated polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 52. The polynucleotide can encode a polypeptide comprising two additional CH2 domains at the N-terminus of an Fc domain linked via a hinge and optionally one or more linkers, wherein the separated polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 52. In some embodiments, the polynucleotide encoding a polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain connected via a hinge and optionally one or more connectors comprises SEQ ID NO: 53.

[0206] In some embodiments, the polynucleotide encodes a polypeptide comprising two separate CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domain. In some embodiments, the “LFLEPS” mutation may comprise the following amino acid substitutions: L234F, L235E, and P331S. In some embodiments, the polynucleotide encodes a polypeptide comprising two separate CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domain, the separate polypeptide comprising SEQ ID NO: 54. The polynucleotide may encode a polypeptide comprising two separate CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domain, the separate polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 54. The polynucleotide may encode a separate polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain connected via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domains, the separate polypeptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 54. In some embodiments, the polynucleotide encoding a separate polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain connected via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domains, comprises SEQ ID NO: 55.

[0207] This polynucleotide can encode any isolated polypeptide disclosed herein and can also encode a second polypeptide, wherein the isolated polypeptide and the second polypeptide share the same amino acid sequence.

[0208] The polynucleotide can encode any isolated polypeptide disclosed herein, which further comprises a CH3 domain. In some embodiments, the polynucleotide encodes a CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. The polynucleotide can encode a CH3 domain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The polynucleotide can encode a CH3 domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the CH3 domain comprises SEQ ID NO: 24 or SEQ ID NO: 179.

[0209] In some embodiments, the polynucleotide encodes an isolated polypeptide comprising a CH3 domain, the isolated polypeptide comprising SEQ ID NO: 106. The polynucleotide may encode an isolated polypeptide comprising a CH3 domain, the isolated polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 106. The polynucleotide may encode an isolated polypeptide comprising a CH3 domain, the isolated polypeptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 106. In some embodiments, the polynucleotide encoding an isolated polypeptide comprising a CH3 domain comprises SEQ ID NO: 105.

[0210] In some embodiments, the polynucleotide encodes a separate polypeptide comprising a CH3 domain, the separate polypeptide comprising SEQ ID NO: 141. The polynucleotide may encode a separate polypeptide comprising a CH3 domain, the separate polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 141. The polynucleotide may encode a separate polypeptide comprising a CH3 domain, the separate polypeptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 141. In some embodiments, the polynucleotide encoding a separate polypeptide comprising a CH3 domain comprises SEQ ID NO: 140.

[0211] This polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, which further comprises Fab. This polynucleotide can encode a Fab comprising a CH1 domain. This polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 17. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 17. The polynucleotide encoding the CH1 domain can comprise SEQ ID NO: 21, SEQ ID NO: 176, or SEQ ID NO: 177. This polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0212] This polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, the isolated polypeptide further comprising Fab. This polynucleotide can encode a Fab comprising a CH1 domain. This polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 100. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 100. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 100. The polynucleotide encoding the CH1 domain can comprise SEQ ID NO: 99. This polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0213] This polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, which further comprises Fab. This polynucleotide can encode a Fab comprising a CH1 domain. This polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 160. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 160. This polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 160. The polynucleotide encoding the CH1 domain can comprise SEQ ID NO: 159. This polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0214] This polynucleotide can encode a Fab that specifically binds to one or more target molecules. The polynucleotide can encode a Fab that specifically binds to perkinin, OCT4, IDO1, FLAG tag, pERK1 / 2, PSD-95, or TIGIT, or the Fab can be a rabbit anti-mouse (RAM) IgG secondary antibody.

[0215] In some embodiments, the polynucleotide may encode a Fab that specifically binds to the perkinin protein. The polynucleotide may encode a Fab that specifically binds to the perkinin protein, the Fab comprising a Fab heavy chain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 4, an HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0216] This polynucleotide encodes a Fab that specifically binds to the perkinin protein, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region containing the amino acid sequence of SEQ ID NO: 7. This polynucleotide can also encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7. Finally, this polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0217] The polynucleotide can encode a Fab that specifically binds to the perkinin protein, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region comprising the amino acid sequence of SEQ ID NO: 9. The polynucleotide can also encode a Fab heavy chain, the Fab heavy chain including a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. Alternatively, the polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9. In some embodiments, the polynucleotide encoding the Fab that specifically binds to the perkinin protein includes SEQ ID NO: 8.

[0218] The polynucleotide can encode a Fab that specifically binds to the perkinin immune system. The Fab contains a Fab light chain, which includes: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 13, an LCDR2 containing the amino acid sequence RAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0219] This polynucleotide encodes a Fab that specifically binds to the perkinin protein, the Fab comprising a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 16. Alternatively, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. Finally, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0220] The polynucleotide can encode a Fab that specifically binds to the perkinin protein, the Fab comprising a Fab light chain, the Fab light chain including a variable region comprising the amino acid sequence of SEQ ID NO: 12. The polynucleotide can encode a Fab light chain, the Fab light chain including a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12. The polynucleotide can encode a Fab light chain, the Fab light chain including a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, in the polynucleotide encoding the Fab that specifically binds to the perkinin protein, the polynucleotide includes SEQ ID NO: 11.

[0221] This polynucleotide encodes a Fab that specifically binds to the perkinin protein. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 4, an HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 6. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 13, an LCDR2 containing the amino acid sequence RAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0222] This polynucleotide encodes a Fab that specifically binds to the perkinin protein. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 7, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 16. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7, and the Fab light chain includes a variable region containing an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 16. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 7, and the Fab light chain includes a variable region containing an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 16.

[0223] This polynucleotide encodes a Fab that specifically binds to the perkinin protein. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 12. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide encoding Fab, which binds to the perkinin immune system specifically, comprises SEQ ID NO: 8 and SEQ ID NO: 11.

[0224] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to the perkinin protein, the Fab comprising the amino acid sequence of SEQ ID NO: 3. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3. In some embodiments, the polynucleotide encoding the Fab that specifically binds to the perkinin protein comprises SEQ ID NO: 2.

[0225] In some embodiments, the polynucleotide may encode a Fab that specifically binds to OCT4. This Fab may encode a Fab that specifically binds to OCT4, the Fab comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 29, an HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 31.

[0226] This polynucleotide encodes a Fab that specifically binds to OCT4, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region containing the amino acid sequence of SEQ ID NO: 32. This polynucleotide can also encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32. Finally, this polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32.

[0227] The polynucleotide can encode a Fab that specifically binds to OCT4, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region comprising the amino acid sequence of SEQ ID NO: 34. The polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34. The polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34. In some embodiments, in the polynucleotide encoding a Fab that specifically binds to OCT4, the polynucleotide includes SEQ ID NO: 33.

[0228] The polynucleotide can encode a Fab that specifically binds to OCT4 immunity, the Fab comprising a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 37, an LCDR2 containing the amino acid sequence GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0229] This polynucleotide encodes a Fab that specifically binds to OCT4, the Fab comprising a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 40. Alternatively, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. Finally, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40.

[0230] The polynucleotide may encode a Fab that specifically binds to OCT4, the Fab comprising a Fab light chain containing the amino acid sequence of SEQ ID NO: 36. The polynucleotide may also encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36. Alternatively, the polynucleotide may encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 36. In some embodiments, the polynucleotide encoding the Fab that specifically binds to OCT4 may contain SEQ ID NO: 35.

[0231] This polynucleotide encodes a Fab that specifically binds to OCT4 immunity. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 29, an HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 31. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 37, an LCDR2 containing the amino acid sequence GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0232] This polynucleotide encodes a Fab that specifically binds to OCT4 immunity. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 32, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 40. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 32, and the Fab light chain includes a variable region containing an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 40. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 32, and the Fab light chain includes a variable region containing an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 40.

[0233] This polynucleotide encodes a Fab that specifically binds to OCT4 immunity. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 36. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 36. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 36. In some embodiments, the polynucleotide encoding Fab that specifically binds to OCT4 immunity comprises SEQ ID NO:33 and SEQ ID NO:35.

[0234] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to OCT4, the Fab comprising the amino acid sequence of SEQ ID NO: 28. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 28. In some embodiments, the polynucleotide encoding the Fab that specifically binds to OCT4 comprises SEQ ID NO: 27.

[0235] In some embodiments, the polynucleotide may encode a Fab that specifically binds to IDO1. The polynucleotide may encode a Fab that specifically binds to IDO1, the Fab comprising a Fab heavy chain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 66, an HCDR2 containing the amino acid sequence of SEQ ID NO: 67, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 68.

[0236] This polynucleotide can encode a Fab that specifically binds to IDO1, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region containing the amino acid sequence of SEQ ID NO: 64. This polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 64. This polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 64.

[0237] The polynucleotide can encode an immune-specific binding Fab of IDO1, the Fab comprising a Fab light chain, the Fab light chain comprising: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 72, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 73.

[0238] This polynucleotide can encode an immune-specific binding Fab to IDO1, the Fab comprising a Fab light chain, the Fab light chain including a variable region containing the amino acid sequence of SEQ ID NO: 71. This polynucleotide can encode a Fab light chain, the Fab light chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. This polynucleotide can encode a Fab light chain, the Fab light chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0239] This polynucleotide encodes a Fab that specifically binds to IDO1, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 66, an HCDR2 containing the amino acid sequence of SEQ ID NO: 67, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 68. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 72, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 73.

[0240] This polynucleotide encodes a Fab that specifically binds to IDO1, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 65, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 71. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71. In some embodiments, the polynucleotide encoding Fab that specifically binds to IDO1 includes SEQ ID NO: 64 and SEQ ID NO: 70.

[0241] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to IDO1, the Fab comprising the amino acid sequence of SEQ ID NO: 79. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 79. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 79. In some embodiments, the polynucleotide encoding the Fab that specifically binds to IDO1 comprises SEQ ID NO: 78.

[0242] This polynucleotide can encode a Fab that specifically binds to a FLAG tag, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region containing the amino acid sequence of SEQ ID NO: 162. This polynucleotide can also encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162. Finally, this polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162.

[0243] The polynucleotide can encode an immune-specific FLAG tag-binding Fab, which contains a Fab light chain comprising: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 168, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 169.

[0244] This polynucleotide can encode an immune-specific FLAG tag-binding Fab, which comprises a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 167. This polynucleotide can also encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167. Finally, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167.

[0245] The polynucleotide encodes a Fab that specifically binds to the FLAG tag. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 163, an HCDR2 containing the amino acid sequence of SEQ ID NO: 164, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 165. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 168, an LCDR2 containing the amino acid sequence of YAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 169.

[0246] This polynucleotide encodes a Fab that specifically binds to a FLAG tag. The Fab comprises a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 162, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 167. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167. In some embodiments, the polynucleotide encoding Fab that binds to the FLAG tag immune-specifically comprises SEQ ID NO: 181 and SEQ ID NO: 166.

[0247] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to a FLAG tag, the Fab comprising the amino acid sequence of SEQ ID NO: 175. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 175. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 175. In some embodiments, in the polynucleotide encoding a Fab that specifically binds to a FLAG tag, the polynucleotide comprises SEQ ID NO: 174.

[0248] In some embodiments, the polynucleotide may encode a Fab as a rabbit anti-mouse IgG secondary antibody (RAM). The polynucleotide may serve as the Fab of the RAM, the Fab comprising a Fab heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129.

[0249] The polynucleotide can encode a Fab as RAM, the Fab comprising a Fab heavy chain, the Fab heavy chain comprising a variable region containing the amino acid sequence of SEQ ID NO: 126. The polynucleotide can encode a Fab heavy chain, the Fab heavy chain comprising a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126. The polynucleotide can encode a Fab heavy chain, the Fab heavy chain comprising a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126.

[0250] The polynucleotide can serve as the Fab of RAM, which contains a Fab light chain comprising: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, an LCDR2 containing the amino acid sequence of GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 133.

[0251] The polynucleotide can encode a Fab as RAM, the Fab comprising a Fab light chain, the Fab light chain comprising a variable region containing the amino acid sequence of SEQ ID NO: 131. The polynucleotide can encode a Fab light chain, the Fab light chain comprising a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 131. The polynucleotide can encode a Fab light chain, the Fab light chain comprising a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 131.

[0252] The polynucleotide can encode a Fab as RAM, the Fab comprising a Fab heavy chain and a Fab light chain, the Fab heavy chain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 127, an HCDR2 containing the amino acid sequence of SEQ ID NO: 128, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 129, the Fab light chain comprising: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, an LCDR2 containing the amino acid sequence of GAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 133.

[0253] This polynucleotide can encode a Fab as RAM, the Fab comprising a Fab heavy chain and a Fab light chain, the Fab heavy chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 126, and the Fab light chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 131. This polynucleotide can encode a Fab heavy chain and a Fab light chain, the Fab heavy chain comprising a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126, and the Fab light chain comprising a variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 131. This polynucleotide can encode a Fab heavy chain and a Fab light chain, the Fab heavy chain comprising a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126, and the Fab light chain comprising a variable region comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 131. In some embodiments, the polynucleotide encoding Fab as RAM comprises SEQ ID NO: 125 and SEQ ID NO: 130.

[0254] In some embodiments, the polynucleotide may encode an antibody comprising a Fab as a RAM, the Fab comprising the amino acid sequence of SEQ ID NO: 139. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 139. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 139. In some embodiments, the polynucleotide encoding the Fab as a RAM comprises SEQ ID NO: 138.

[0255] In some embodiments, the polynucleotide may encode a Fab that specifically binds to pERK1 / 2. This polynucleotide may encode a Fab that specifically binds to pERK1 / 2, the Fab comprising a Fab heavy chain containing: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0256] This polynucleotide encodes a Fab that specifically binds to pERK1 / 2, the Fab comprising a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 84. This polynucleotide can also encode a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84. Finally, this polynucleotide can encode a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84.

[0257] This polynucleotide can encode an immune-specific binding Fab of pERK1 / 2, the Fab comprising a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, an LCDR2 containing the amino acid sequence of KVS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0258] This polynucleotide can encode an immune-specific Fab that binds to pERK1 / 2, the Fab comprising a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 90. Alternatively, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. Finally, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0259] This polynucleotide encodes a Fab that specifically binds to pERK1 / 2, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 85, an HCDR2 containing the amino acid sequence of SEQ ID NO: 86, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 87. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, an LCDR2 containing the amino acid sequence of KVS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0260] This polynucleotide encodes a Fab that specifically binds to pERK1 / 2, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 84, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 90. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90. In some embodiments, the polynucleotide encoding Fab that binds specifically to pERK1 / 2 includes SEQ ID NO: 83 and SEQ ID NO: 89.

[0261] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to pERK1 / 2, the Fab comprising the amino acid sequence of SEQ ID NO: 98. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 98. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 98. In some embodiments, the polynucleotide encoding the Fab that specifically binds to pERK1 / 2 comprises SEQ ID NO: 97.

[0262] In some embodiments, the polynucleotide may encode a Fab that specifically binds to PSD-95. The polynucleotide may encode a Fab that specifically binds to PSD-95, the Fab comprising a Fab heavy chain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 111, an HCDR2 containing the amino acid sequence of SEQ ID NO: 112, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 113.

[0263] This polynucleotide can encode a Fab that specifically binds to PSD-95, the Fab comprising a Fab heavy chain, the Fab heavy chain including a variable region containing the amino acid sequence of SEQ ID NO: 110. This polynucleotide can also encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110. Finally, this polynucleotide can encode a Fab heavy chain, the Fab heavy chain including a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110.

[0264] The polynucleotide can encode an immune-specific binding Fab to PSD-95, the Fab comprising a Fab light chain containing: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116, an LCDR2 containing the amino acid sequence of DTS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 117.

[0265] This polynucleotide can encode an immune-specific binding Fab to PSD-95, the Fab comprising a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 115. This polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. This polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115.

[0266] This polynucleotide encodes a Fab that specifically binds to PSD-95, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 111, an HCDR2 containing the amino acid sequence of SEQ ID NO: 112, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 113. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116, an LCDR2 containing the amino acid sequence of DTS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 117.

[0267] This polynucleotide encodes a Fab that specifically binds to PSD-95, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 110, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 115. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115. In some embodiments, the polynucleotide encoding Fab that specifically binds to PSD-95 immunity comprises SEQ ID NO: 125 and SEQ ID NO: 130.

[0268] In some embodiments, the polynucleotide may encode an antibody comprising a Fab that specifically binds to PSD-95, the Fab comprising the amino acid sequence of SEQ ID NO: 123. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 123. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 123. In some embodiments, the polynucleotide encoding the Fab that specifically binds to PSD-95 comprises SEQ ID NO: 122.

[0269] In some embodiments, the polynucleotide may encode a Fab that specifically binds to TIGIT. This Fab may encode a Fab that specifically binds to TIGIT, the Fab comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 146, an HCDR2 containing the amino acid sequence of SEQ ID NO: 147, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 148.

[0270] This polynucleotide encodes a Fab that specifically binds to TIGIT, the Fab comprising a Fab heavy chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 145. This polynucleotide can also encode a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145. Finally, this polynucleotide can encode a Fab heavy chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145.

[0271] The polynucleotide can encode a Fab that specifically binds to TIGIT, the Fab comprising a Fab light chain containing: a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151, an LCDR2 containing the amino acid sequence of SAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 152.

[0272] This polynucleotide can encode a Fab that specifically binds to TIGIT, the Fab comprising a Fab light chain containing a variable region comprising the amino acid sequence of SEQ ID NO: 150. This polynucleotide can also encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. Finally, this polynucleotide can encode a Fab light chain containing a variable region comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0273] This polynucleotide encodes a Fab that specifically binds to TIGIT, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain comprises: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 146, an HCDR2 containing the amino acid sequence of SEQ ID NO: 147, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 148. The Fab light chain comprises: a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151, an LCDR2 containing the amino acid sequence of SAS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 152.

[0274] This polynucleotide encodes a Fab that specifically binds to TIGIT, comprising a Fab heavy chain and a Fab light chain. The Fab heavy chain includes a variable region containing the amino acid sequence of SEQ ID NO: 145, and the Fab light chain includes a variable region containing the amino acid sequence of SEQ ID NO: 150. Alternatively, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145, and the Fab light chain includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. Finally, this polynucleotide can encode a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145, and the Fab light chain includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150. In some embodiments, the polynucleotide encoding Fab that binds to TIGIT immune-specifically comprises SEQ ID NO: 144 and SEQ ID NO: 149.

[0275] In some embodiments, the polynucleotide may encode a Fab that specifically binds to TIGIT, the Fab comprising the amino acid sequence of SEQ ID NO: 158. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 158. The polynucleotide may encode a Fab comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 158. In some embodiments, the polynucleotide encoding the Fab that specifically binds to TIGIT comprises SEQ ID NO: 157.

[0276] This polynucleotide can encode any antibody disclosed herein, wherein the antibody has isotype IgG.

[0277] This document discloses vectors comprising any of the nucleic acid molecules described herein, which encode isolated polypeptides or antibodies. It also discloses host cells comprising any of the vectors disclosed herein. Suitable host cells include eukaryotic and prokaryotic cells.

[0278] This document discloses methods for producing isolated peptides or antibodies, which include culturing host cells containing any of the vectors disclosed herein under conditions suitable for expressing the isolated peptides or antibodies, and purifying the isolated peptides or antibodies.

[0279] This article discloses an antibody comprising an isolated polypeptide linked to a Fab domain, the isolated polypeptide comprising the amino acid sequence of formula I: Z-(XY) n -Z r ) p -X, where Z is the hinge region, X is the CH2 structural domain, Y is the joint, n is an integer of 1 or greater, p is an integer of 2 or greater, and r is an integer of 0 or 1.

[0280] The hinge (Z) region may contain the amino acid sequence of SEQ ID NO: 18. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. The hinge (Z) may contain the amino acid sequence of SEQ ID NO: 48. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. The hinge (Z) may contain the amino acid sequence of SEQ ID NO: 102. The hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0281] The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 19. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 49. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. The CH2 domain (X) may contain the amino acid sequence of SEQ ID NO: 104. The CH2 domain (X) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The CH2 domain (X) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0282] In some embodiments, the antibody comprising the isolated polypeptide may include one or more adapters (Y) comprising the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The one or more adapters (Y) may comprise GS. The one or more adapters (Y) may comprise GGS. The one or more adapters (Y) may comprise SEQ ID NO: 14. The one or more adapters (Y) may comprise SEQ ID NO: 38. The one or more adapters may comprise SEQ ID NO: 43. The one or more adapters (Y) may comprise a sequence having at least 90% sequence identity with SEQ ID NO: 43. The one or more adapters (Y) may comprise SEQ ID NO: 44. The one or more adapters (Y) may comprise a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0283] The isolated polypeptide can contain one linker (Y) such that n is 1. The isolated polypeptide can contain two linkers (Y) such that n is 2. The isolated polypeptide can contain three linkers (Y) such that n is 3. The isolated polypeptide can contain four linkers (Y) such that n is 4. The isolated polypeptide can contain five linkers (Y) such that n is 5.

[0284] In some embodiments, the antibody comprising the isolated polypeptide may contain two or more CH2-connector regions ((XY)). n ) p The region contains two CH2-linker regions ((XY)) such that p is 2 or greater. The isolated polypeptide may contain two CH2-linker regions ((XY)). n ) p ), making p = 2. The isolated polypeptide can contain three CH2-linker regions ((XY) n ) p ), making p = 3. The isolated polypeptide can contain four CH2-linker regions ((XY) n ) p ), making p = 4. The isolated polypeptide can contain five CH2-linker regions ((XY) n ) p ), making p = 5.

[0285] In some embodiments, the antibody comprising the isolated polypeptide may contain two or more CH2-linker-hinge regions ((XY) n -Z r ) p The region contains two CH2-linker-hinge regions ((XY)) such that p is 2 or greater. The isolated polypeptide may contain two CH2-linker-hinge regions ((XY)). n-Z r ) p ), making p = 2. The isolated polypeptide can contain three CH2-linker-hinge regions ((XY)). n -Z r ) p ), making p = 3. The isolated polypeptide can contain four CH2-linker-hinge regions ((XY) n -Z r ) p ), making p = 4. The isolated polypeptide can contain five CH2-linker-hinge regions ((XY) n -Z r ) p ), making p = 5.

[0286] In some embodiments, the antibody comprising the isolated polypeptide contains (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 18, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 19, Y (linker) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0287] In some embodiments, the antibody comprising the isolated polypeptide contains (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 48, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 49, Y (connector) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0288] In some embodiments, the antibody comprising the isolated polypeptide contains (XY) n -Z r ) p It is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 104, Y (connector) contains the amino acid sequence of SEQ ID NO: 44, wherein n is 1, r is 0 or 1, and p is 2.

[0289] In some embodiments, the antibody comprising the isolated polypeptide contains (XY) n -Z r ) pIt is essentially composed of or consists of, wherein Z (hinge) contains the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) contains the amino acid sequence of SEQ ID NO: 104, Y (linker) contains the amino acid sequence of SEQ ID NO: 45, wherein n is 1, r is 0 or 1, and p is 2.

[0290] In some embodiments, the antibody comprising the isolated polypeptide comprises two additional CH2 domains at the N-terminus of the Fc domain linked via a hinge and optionally one or more adapters. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain linked via a hinge and optionally one or more adapters may comprise the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated polypeptide comprising the CH3 domain comprises SEQ ID NO: 52. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain linked via a hinge and optionally one or more adapters may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 52. The isolated polypeptide comprising two additional CH2 domains at the N-terminus of the Fc domain linked via a hinge and optionally one or more adapters may comprise an amino acid sequence having at least 95% identity with SEQ ID NO: 52.

[0291] In some embodiments, the antibody comprising the isolated polypeptide comprises two additional CH2 domains at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and includes an “LFLEPS” mutation within the lower hinge and CH2 domains. The “LFLEPS” mutation may comprise the following amino acid substitutions: L234F, L235E, and P331S. The isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and including an “LFLEPS” mutation within the lower hinge and CH2 domain, comprises SEQ ID NO: 54. The isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain linked via a hinge and optionally one or more linkers, and including an “LFLEPS” mutation within the lower hinge and CH2 domain, may comprise an amino acid sequence having at least 90% identity with SEQ ID NO: 54. An isolated polypeptide comprising two additional CH2 structures at the N-terminus of the Fc domain connected via a hinge and optionally one or more linkers, and containing an “LFLEPS” mutation within the lower hinge and CH2 domain, may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 54.

[0292] In some embodiments, the antibody comprising the isolated peptide disclosed herein has a background signal reduction of at least 1 / 3 compared to the corresponding wild-type antibody. In some embodiments, the background signal reduction is at least 1 / 3, at least 1 / 2, at least 3 / 5, at least 2 / 3, at least 5 / 7, at least 3 / 4, at least 7 / 9, or at least 4 / 5 compared to the corresponding wild-type antibody.

[0293] In some embodiments, the antibody comprising any isolated peptide disclosed herein has at least a 0.5-fold increase in sensitivity or signal enhancement compared to the corresponding wild-type antibody. In some embodiments, the increase in sensitivity is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, or at least 9-fold compared to the corresponding wild-type antibody.

[0294] This document discloses antibodies that specifically bind to the perkinin protein, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 13, an LCDR2 comprising the amino acid sequence RAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to the perkinin also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0295] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 50. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0296] In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0297] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0298] In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48.

[0299] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0300] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0301] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0302] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 3 and a light chain having the polypeptide sequence of SEQ ID NO: 12. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 12.

[0303] In some embodiments, the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 2 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 11.

[0304] This document discloses antibodies that specifically bind to OCT4, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to OCT4 also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0305] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 50. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0306] In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0307] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the first hinge and the second hinge each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0308] In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48.

[0309] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0310] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0311] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 32 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40.

[0312] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 28 and a light chain having the polypeptide sequence of SEQ ID NO: 36. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 28 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 36.

[0313] In some embodiments, the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 27 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 35.

[0314] This document discloses antibodies that specifically bind to IDO1, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 66, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 68, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to IDO1 also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0315] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0316] In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0317] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0318] In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48.

[0319] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0320] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0321] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 65 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 71. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0322] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 75 and a light chain having the polypeptide sequence of SEQ ID NO: 77. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 75 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 77. The antibody may also comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 75 and a light chain having at least 95% sequence identity with SEQ ID NO: 77.

[0323] In some embodiments, the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 74 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 76.

[0324] This document discloses antibodies that specifically bind to FLAG tags, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 163, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 164, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 168, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 169, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to the FLAG tag also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0325] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0326] In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0327] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0328] In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48.

[0329] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0330] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0331] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 167. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167.

[0332] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 171 and a light chain having the polypeptide sequence of SEQ ID NO: 173. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 171 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 173. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 171 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 173.

[0333] In some embodiments, the antibody comprises a heavy chain encoded by a polynucleotide sequence of SEQ ID NO: 170 and a light chain encoded by a polynucleotide sequence of SEQ ID NO: 172.

[0334] This article discloses rabbit anti-mouse IgG secondary antibodies (RAMs) comprising heavy chains (HC) and light chains (LC) paired to form a binding domain, substantially composed of heavy chains and light chains or composed of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 133, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some implementations, the rabbit anti-mouse IgG secondary antibody also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0335] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0336] In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0337] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0338] In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48.

[0339] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0340] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 141. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 141. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 141.

[0341] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 131. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 131. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 131.

[0342] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 135 and a light chain having the polypeptide sequence of SEQ ID NO: 137. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 135 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 137. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 135 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 137.

[0343] In some embodiments, the antibody comprises a heavy chain encoded by a polynucleotide sequence of SEQ ID NO: 134 and a light chain encoded by a polynucleotide sequence of SEQ ID NO: 136.

[0344] This document discloses antibodies that specifically bind to pERK1 / 2, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 91, an LCDR2 comprising the amino acid sequence of KVS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to pERK1 / 2 also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0345] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 104. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 104. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0346] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 102. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 102. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0347] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0348] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 106. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 106. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 106.

[0349] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 84 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0350] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 94 and a light chain having the polypeptide sequence of SEQ ID NO: 96. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 94 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 96. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 94 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 96.

[0351] In some embodiments, the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 93 and a light chain encoded by the polynucleotide sequence of SEQ ID NO: 95.

[0352] This document discloses antibodies that specifically bind to PSD-95, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 111, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 112, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 113, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence of DTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 117, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to PSD-95 also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0353] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 104. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 104. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0354] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 102. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 102. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0355] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0356] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 106. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 106. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 106.

[0357] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 115. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115.

[0358] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 119 and a light chain having the polypeptide sequence of SEQ ID NO: 121. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 119 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 121. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 119 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 121.

[0359] In some embodiments, the antibody comprises a heavy chain encoded by a polynucleotide sequence of SEQ ID NO: 118 and a light chain encoded by a polynucleotide sequence of SEQ ID NO: 120.

[0360] This document discloses antibodies that specifically bind to TIGIT, wherein these antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain, consisting substantially of heavy chains and light chains or consisting of heavy chains and light chains, wherein the binding domain comprises: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 148, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 151, an LCDR2 comprising the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 152, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the antibody that specifically binds to TIGIT also includes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0361] The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first CH2 domain, the second CH2 domain, and the third CH2 domain each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0362] The first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first hinge and the second hinge may each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge may each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0363] The first and second connectors may each contain the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second connectors may contain GS. The first and second connectors may contain GGS. The first and second connectors may contain SEQ ID NO: 14. The first and second connectors may contain SEQ ID NO: 38. The first and second connectors may contain SEQ ID NO: 43. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 43. The first and second connectors may contain SEQ ID NO: 44. The first and second connectors may contain a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0364] In some embodiments, the antibody comprises a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may also comprise a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. Alternatively, the antibody may comprise a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0365] In some embodiments, the antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145 and a light chain variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. The antibody may comprise a heavy chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145 and a light chain variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0366] In some embodiments, the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 154 and a light chain having the polypeptide sequence of SEQ ID NO: 156. The antibody may comprise a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 154 and a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 156. The antibody may comprise a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 154 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 156.

[0367] In some embodiments, the antibody comprises a heavy chain encoded by a polynucleotide sequence of SEQ ID NO: 153 and a light chain encoded by a polynucleotide sequence of SEQ ID NO: 155.

[0368] In some embodiments, the antibody is labeled. The label can be selected from fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, polymer dyes, particles, electron transfer agents, haptens, enzymes, radioisotopes, chromophores, and oligonucleotide-based fluorescent dyes. The label can be a fluorophore. The label can be a fluorescent protein. The label can be a phosphorescent dye. The label can be a tandem dye. The label can be a polymer dye. The label can be a particle. The label can be an electron transfer agent. The label can be a hapten. The label can be an enzyme. The label can be a radioisotope. The label can be a chromophore. The label can be an oligonucleotide-based fluorescent dye.

[0369] In some implementations, the marker is a fluorophore. The fluorophore can be pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzoindole, oxazole or benzoxazole, thiazole or benzothiazole, indole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), anthocyanin, carbonyl anthocyanin, benzoanthocyanin, quinolone, porphyrin, salicylates, anthraquinones, azulene, perylene, pyridine, quinoline, boron polyacrylamide, xanthannadenine, rhodamine, silanolamine, fluorescein, p-methylaminophenol, rhodamine, oxazine or benzoxazine, benzophenoxazine, carbazine, finasteride, coumarin, furan or benzofuran, benzyphenadenone, benzopyranone, styraxene, squaricine, halogen, anthraquinone, quinazolinone, oligonucleotide-based fluorescent dyes, conjugated polymer dyes, or tandem dyes. The fluorophore can be pyrene. The fluorophore can be anthracene. The fluorophore can be naphthalene. The fluorophore can be acridine. The fluorophore can be stilbene. The fluorophore can be indole. The fluorophore can be benzoindole. The fluorophore can be oxazole. The fluorophore can be benzoxazole. The fluorophore can be thiazole. The fluorophore can be benzothiazole. The fluorophore can be indole. The fluorophore can be 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD). The fluorophore can be anthocyanin. The fluorophore can be carbonyl anthocyanin. The fluorophore can be benzoanthocyanin. The fluorophore can be aquinone. The fluorophore can be porphyrin. The fluorophore can be salicylate. The fluorophore can be anthraquinone. The fluorophore can be azulene. The fluorophore can be perylene. The fluorophore can be pyridine. The fluorophore can be quinoline. The fluorophore can be boron polyacrylamide. The fluorophore can be xanthan. The fluorophore can be rhodamine. The fluorophore can be silyl rhodamine. The fluorophore can be fluorescein. The fluorophore can be p-aminophenol. The fluorophore can be roxamine. The fluorophore can be an oxazine. The fluorophore can be benzoxazine. The fluorophore can be benzophenoxazine. The fluorophore can be carbazine. The fluorophore can be finasterone. The fluorophore can be coumarin. The fluorophore can be furan. The fluorophore can be benzofuran. The fluorophore can be benzyphenanone. The fluorophore can be benzopyranone. The fluorophore can be styraquinone. The fluorophore can be squaricine. The fluorophore can be halogen. The fluorophore can be anthraquinone. The fluorophore can be quinazolinone. The fluorophore can be an oligonucleotide-based fluorescent dye. The fluorophore can be a conjugated polymer dye. The fluorophore can be a tandem dye.

[0370] In some implementations, the marker is a particle. The particle can be a semiconductor nanocrystal.

[0371] In some implementations, the label is a hapten. A hapten can be biotin or a biotin derivative. A hapten can be biotin. A hapten can be a biotin derivative.

[0372] In some implementations, the label is "enzyme." The enzyme can be a peroxidase, phosphatase, glycosidase, or luciferase. The enzyme can be a peroxidase. The enzyme can be a phosphatase. The enzyme can be a glycosidase. The enzyme can be a luciferase.

[0373] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of polynucleotides encoding antibodies that specifically bind to the perkinin protein, an antibody comprising a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 13, an LCDR2 comprising the amino acid sequence RAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0374] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 23.

[0375] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may also encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, among the polynucleotides encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain comprises SEQ ID NO: 62.

[0376] The polynucleotide may encode a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0377] In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide also encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Alternatively, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, of the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0378] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0379] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0380] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0381] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 3 and a light chain having the polypeptide sequence of SEQ ID NO: 12. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 and the light chain having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 2 and SEQ ID NO: 11.

[0382] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of polynucleotides encoding an antibody that specifically binds to OCT4, an antibody comprising a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0383] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 23.

[0384] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may also encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, among the polynucleotides encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain comprises SEQ ID NO: 62.

[0385] The polynucleotide may encode a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0386] In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide also encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Alternatively, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, of the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0387] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0388] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 32 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40.

[0389] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0390] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 28 and a light chain having the polypeptide sequence of SEQ ID NO: 36. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 28, and the light chain having at least 95% sequence identity with SEQ ID NO: 36. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 27 and SEQ ID NO: 35.

[0391] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of polynucleotides encoding antibodies that specifically bind to IDO1, an antibody comprising a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 66, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 68, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and an optional first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge. In some implementations, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0392] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide comprises SEQ ID NO: 178.

[0393] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may also encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, among the polynucleotides encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain comprises SEQ ID NO: 62.

[0394] The polynucleotide may encode a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0395] In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide also encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Alternatively, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, of the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0396] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0397] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0398] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 65 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 71. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0399] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 75 and a light chain having the polypeptide sequence of SEQ ID NO: 77. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 75, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 77. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 75, and the light chain having at least 95% sequence identity with SEQ ID NO: 77. In some embodiments, in the polynucleotide encoding the heavy chain and light chain, the polynucleotide comprises both SEQ ID NO: 75 and SEQ ID NO: 77.

[0400] This document discloses nucleic acid molecules comprising, substantially comprising, or composed of, polynucleotides encoding antibodies that specifically bind to FLAG tags, an antibody comprising a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 163, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 164, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 168, an LCDR2 comprising the amino acid sequence of YAS, and a light chain complementarity-determining region 1 comprising the amino acid sequence of SEQ ID NO: 168. LCDR3 has an amino acid sequence of 169, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some embodiments, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0401] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 23.

[0402] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may also encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, among the polynucleotides encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain comprises SEQ ID NO: 62.

[0403] The polynucleotide may encode a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0404] In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide also encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Alternatively, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, of the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0405] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0406] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0407] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 167. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167.

[0408] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 171 and a light chain having the polypeptide sequence of SEQ ID NO: 173. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 171, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 173. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 171, and the light chain having at least 95% sequence identity with SEQ ID NO: 173. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 170 and SEQ ID NO: 172.

[0409] This document discloses nucleic acid molecules containing, substantially composed of, or composed of polynucleotides encoding rabbit anti-mouse IgG secondary antibodies (RAM), wherein these antibodies contain a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 127, an HCDR2 containing the amino acid sequence of SEQ ID NO: 128, an HCDR3 containing the amino acid sequence of SEQ ID NO: 129, a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, an LCDR2 containing the amino acid sequence of GAS, and a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132. LCDR3 is a polynucleotide with an amino acid sequence of 133, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some embodiments, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0410] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 49. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 23.

[0411] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 49. The polynucleotide may also encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. Alternatively, the polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, among the polynucleotides encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain comprises SEQ ID NO: 62.

[0412] The polynucleotide may encode a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0413] In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide also encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Alternatively, the polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, of the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0414] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0415] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0416] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 131. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 131. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 131.

[0417] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 135 and a light chain having the polypeptide sequence of SEQ ID NO: 137. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 135, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 137. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 135, and the light chain having at least 95% sequence identity with SEQ ID NO: 137. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 134 and SEQ ID NO: 136.

[0418] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of, polynucleotides encoding antibodies that specifically bind to pERK1 / 2, an antibody comprising a heavy chain (HC) and a light chain (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 91, an LCDR2 comprising the amino acid sequence of KVS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92, and wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some implementations, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0419] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 104. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 103.

[0420] The polynucleotide may encode a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 102. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102. In some embodiments, in the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 101.

[0421] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0422] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 106.

[0423] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 84 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 90. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0424] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 94 and a light chain having the polypeptide sequence of SEQ ID NO: 96. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 94, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 96. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 94, and the light chain having at least 95% sequence identity with SEQ ID NO: 96. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 93 and SEQ ID NO: 95.

[0425] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of, polynucleotides encoding antibodies that specifically bind to PSD-95 immunoreactivity. These antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 111; an HCDR2 containing the amino acid sequence of SEQ ID NO: 112; an HCDR3 containing the amino acid sequence of SEQ ID NO: 113; a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116; an LCDR2 containing the amino acid sequence of DTS; and a light chain complementarity-determining region 1 containing the amino acid sequence of SEQ ID NO: 116. LCDR3 has an amino acid sequence of 117, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some embodiments, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0426] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 104. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 103.

[0427] The polynucleotide may encode a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 102. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102. In some embodiments, in the polynucleotides encoding the first and second hinges, the polynucleotide encoding each hinge comprises SEQ ID NO: 101.

[0428] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0429] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 106.

[0430] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 115. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115.

[0431] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 119 and a light chain having the polypeptide sequence of SEQ ID NO: 121. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 119, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 121. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 119, and the light chain having at least 95% sequence identity with SEQ ID NO: 121. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 118 and SEQ ID NO: 120.

[0432] This document discloses nucleic acid molecules comprising, substantially composed of, or composed of, polynucleotides encoding antibodies that specifically bind to TIGIT, an immune response. These antibodies comprise heavy chains (HC) and light chains (LC) paired to form a binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 146; an HCDR2 containing the amino acid sequence of SEQ ID NO: 147; an HCDR3 containing the amino acid sequence of SEQ ID NO: 148; a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151; an LCDR2 containing the amino acid sequence of SEQ ID NO: 151; and a light chain complementarity-determining region 1 containing the amino acid sequence of SEQ ID NO: 151. LCDR3 has an amino acid sequence of 152, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge. In some embodiments, the polynucleotide also encodes a CH3 domain linked to the C-terminus of the third CH2 domain.

[0433] The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising the amino acid sequence of SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide may encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain comprises SEQ ID NO: 23.

[0434] The polynucleotide may encode a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 22.

[0435] The polynucleotide may encode a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the polynucleotide encodes a first hinge and a second hinge, each comprising the amino acid sequence of SEQ ID NO: 48. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotide encodes a first hinge and a second hinge, each comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, in the polynucleotides encoding the first hinge and the second hinge, the polynucleotide encoding each hinge comprises SEQ ID NO: 51.

[0436] This polynucleotide can encode a first and second connector containing the amino acid sequence GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing GS. The first and second connectors can contain GGS. This polynucleotide can encode a first and second connector containing SEQ ID NO: 14. This polynucleotide can encode a first and second connector containing SEQ ID NO: 38. This polynucleotide can encode a first and second connector containing SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. This polynucleotide can encode a first and second connector containing SEQ ID NO: 44. This polynucleotide can encode a first and second connector containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0437] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain, the CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0438] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 145 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150. The polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145, and the light chain variable region has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. Alternatively, the polynucleotide may encode an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145, and the light chain variable region has an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0439] In some embodiments, the polynucleotide encodes an antibody comprising a heavy chain having the polypeptide sequence of SEQ ID NO: 154 and a light chain having the polypeptide sequence of SEQ ID NO: 156. The polynucleotide may encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 154, and the light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 156. The polynucleotide may also encode an antibody comprising a heavy chain and a light chain, the heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 154, and the light chain having at least 95% sequence identity with SEQ ID NO: 156. In some embodiments, the polynucleotide encoding the heavy chain and light chain comprises SEQ ID NO: 153 and SEQ ID NO: 155.

[0440] This document discloses vectors comprising any of the nucleic acid molecules described herein, which contain polynucleotides encoding antibodies. It also discloses host cells comprising any of the vectors disclosed herein. Suitable host cells include eukaryotic and prokaryotic cells.

[0441] The antibodies or peptides according to this disclosure can be produced using any known and well-established expression system and recombinant cell culture technique, for example, by expression in a bacterial host (prokaryotic system) or a eukaryotic system (such as yeast, fungi, insect cells, or mammalian cells). The antibodies or peptides of this disclosure can be produced in transgenic organisms (such as goats or plants). The antibodies or peptides of this disclosure can also be produced by chemical synthesis.

[0442] This document discloses methods for detecting the presence of target proteins in samples, comprising, substantially comprising, or comprising the following: applying any isolated polypeptide or any antibody disclosed herein to a sample containing a target protein; incubating the isolated polypeptide or antibody in the sample under conditions that allow the isolated polypeptide or antibody to bind to the target protein and form bound isolated polypeptide or bound antibody; and determining the presence of bound isolated polypeptide or bound antibody, thereby detecting the presence of the target protein.

[0443] This document discloses methods for detecting the presence of target proteins in samples, comprising, substantially comprising, or comprising the following: applying any isolated polypeptide disclosed herein to a sample containing a target protein; incubating the isolated polypeptide in the sample under conditions that allow the isolated polypeptide to bind to the target protein and form a bound isolated polypeptide; and determining the presence of the bound isolated polypeptide, thereby detecting the presence of the target protein.

[0444] This document discloses methods for detecting the presence of target proteins in samples, which include, consist essentially of, or consist of the following: applying any antibody disclosed herein to a sample containing a target protein, incubating the antibody in the sample under conditions that allow the antibody to bind to the target protein and form a bound antibody, and determining the presence of the bound antibody, thereby detecting the presence of the target protein.

[0445] This document discloses the use of any isolated peptide or antibody disclosed herein in detecting the presence of a target protein in a sample. These uses may include, substantially consist of, or consist of: applying any isolated peptide or antibody disclosed herein to a sample containing a target protein; incubating the isolated peptide or antibody in the sample under conditions that allow the isolated peptide or antibody to bind to the target protein and form a bound isolated peptide or bound antibody; and determining the presence of the bound isolated peptide or bound antibody, thereby detecting the presence of the target protein.

[0446] This document discloses the use of any of the isolated peptides disclosed herein in detecting the presence of a target protein in a sample. These uses may include, consist substantially of, or consist of: applying any of the isolated peptides disclosed herein to a sample containing a target protein, incubating the isolated peptide in the sample under conditions that allow the isolated peptide to bind to the target protein and form a bound isolated peptide, and determining the presence of the bound isolated peptide, thereby detecting the presence of the target protein.

[0447] This document discloses the use of any of the antibodies disclosed herein in detecting the presence of a target protein in a sample. These uses may include, consist essentially of, or consist of: applying any of the antibodies disclosed herein to a sample containing a target protein, incubating the antibody in the sample under conditions that allow the antibody to bind to the target protein and form a bound antibody, and determining the presence of the bound antibody, thereby detecting the presence of the target protein.

[0448] In some embodiments of the method or application, a smaller amount of the isolated peptide or antibody disclosed herein is required relative to the amount of wild-type isolated peptide or wild-type antibody required to generate a detectable signal in an equivalent method. A smaller amount of the isolated peptide disclosed herein may be required relative to the amount of wild-type isolated peptide required to generate a detectable signal in an equivalent method. A smaller amount of the antibody disclosed herein may be required relative to the amount of wild-type antibody required to generate a detectable signal in an equivalent method. The isolated peptides and antibodies disclosed herein provide enhanced sensitivity at lower concentrations, which allows for the generation of robust signals at concentrations where wild-type isolated peptides or wild-type antibodies would provide less signal.

[0449] In some implementations of the method or application, a smaller amount of sample (e.g., rare clinical sample) is used compared to an equivalent method using wild-type isolated peptides or wild-type antibodies, but still produces a stronger signal.

[0450] In some embodiments of the method or application, it is possible to detect a lower concentration of the target protein in a sample compared to an equivalent method using a wild-type isolated peptide or wild-type antibody.

[0451] In some embodiments of the method or use, the isolated peptides or antibodies disclosed herein bind less to the background of cells expressing Fcγ receptor (FcγR) compared to wild-type isolated peptides or wild-type antibodies.

[0452] In some embodiments of the method or use, the target protein is OCT4, pergenin, IDO1, a FLAG tag, pERK1 / 2, PSD-95, or TIGIT. In some embodiments of the method or use, the target protein is OCT4. In some embodiments of the method or use, the target protein is pergenin. In some embodiments of the method or use, the target protein is IDO1. In some embodiments of the method or use, the target protein is a FLAG tag. In some embodiments of the method or use, the target protein is pERK1 / 2. In some embodiments of the method or use, the target protein is PSD-95. In some embodiments of the method or use, the target is TIGIT.

[0453] Target proteins may be present in low abundance in a sample. Low abundance targets are expressed or present at low levels below the detection limit, or produce a very weak signal when used with wild-type antibodies in standard immunoassays and immunoassays. Low abundance includes situations where the target protein cannot be detected or is difficult to detect in specific assays using wild-type antibodies.

[0454] In some embodiments of the method or use, the presence of a bound isolated peptide or bound antibody is determined by applying a secondary antibody to the sample, wherein the secondary antibody is labeled. In some embodiments of the method, the secondary antibody is the rabbit anti-mouse (RAM) of this disclosure.

[0455] In some embodiments of the method or use, the antibody is labeled. The label can be selected from fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, polymer dyes, particles, electron transfer agents, haptens, enzymes, radioisotopes, chromophores, and oligonucleotide-based fluorescent dyes. The label can be a fluorophore. The label can be a fluorescent protein. The label can be a phosphorescent dye. The label can be a tandem dye. The label can be a polymer dye. The label can be a particle. The label can be an electron transfer agent. The label can be a hapten. The label can be an enzyme. The label can be a radioisotope. The label can be a chromophore. The label can be an oligonucleotide-based fluorescent dye.

[0456] In some implementations, the marker is a fluorophore. The fluorophore can be pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzoindole, oxazole or benzoxazole, thiazole or benzothiazole, indole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), anthocyanin, carbonyl anthocyanin, benzoanthocyanin, quinolone, porphyrin, salicylates, anthraquinones, azulene, perylene, pyridine, quinoline, boron polyacrylamide, xanthannadenine, rhodamine, silanolamine, fluorescein, p-methylaminophenol, rhodamine, oxazine or benzoxazine, benzophenoxazine, carbazine, finasteride, coumarin, furan or benzofuran, benzyphenadenone, benzopyranone, styraxene, squaricine, halogen, anthraquinone, quinazolinone, oligonucleotide-based fluorescent dyes, conjugated polymer dyes, or tandem dyes. The fluorophore can be pyrene. The fluorophore can be anthracene. The fluorophore can be naphthalene. The fluorophore can be acridine. The fluorophore can be stilbene. The fluorophore can be indole. The fluorophore can be benzoindole. The fluorophore can be oxazole. The fluorophore can be benzoxazole. The fluorophore can be thiazole. The fluorophore can be benzothiazole. The fluorophore can be indole. The fluorophore can be 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD). The fluorophore can be anthocyanin. The fluorophore can be carbonyl anthocyanin. The fluorophore can be benzoanthocyanin. The fluorophore can be aquinone. The fluorophore can be porphyrin. The fluorophore can be salicylate. The fluorophore can be anthraquinone. The fluorophore can be azulene. The fluorophore can be perylene. The fluorophore can be pyridine. The fluorophore can be quinoline. The fluorophore can be boron polyacrylamide. The fluorophore can be xanthan. The fluorophore can be rhodamine. The fluorophore can be silyl rhodamine. The fluorophore can be fluorescein. The fluorophore can be p-aminophenol. The fluorophore can be roxamine. The fluorophore can be an oxazine. The fluorophore can be benzoxazine. The fluorophore can be benzophenoxazine. The fluorophore can be carbazine. The fluorophore can be finasterone. The fluorophore can be coumarin. The fluorophore can be benzofuran. The fluorophore can be benzyphenone. The fluorophore can be benzopyranone. The fluorophore can be styraquinone. The fluorophore can be squaricine. The fluorophore can be halogen. The fluorophore can be anthraquinone. The fluorophore can be quinazolinone. The fluorophore can be an oligonucleotide-based fluorescent dye. The fluorophore can be a conjugated polymer dye. The fluorophore can be a tandem dye.

[0457] In some implementations, the marker is a particle. The particle can be a semiconductor nanocrystal.

[0458] In some implementations, the label is a hapten. A hapten can be biotin or a biotin derivative. A hapten can be biotin. A hapten can be a biotin derivative.

[0459] In some implementations, the label is "enzyme." The enzyme can be a peroxidase, phosphatase, glycosidase, or luciferase. The enzyme can be a peroxidase. The enzyme can be a phosphatase. The enzyme can be a glycosidase. The enzyme can be a luciferase.

[0460] These methods or applications may also include adding an enzyme substrate selected from chromogenic, fluorescent, and chemiluminescent substrates. The enzyme substrate may be a chromogenic substrate. The enzyme substrate may be a fluorescent substrate. The enzyme substrate may be a chemiluminescent substrate.

[0461] These methods or uses may include detecting labeled antibodies using flow cytometry, immunohistochemistry, immunocytochemistry, or Western blotting. Labeled antibodies can be detected using flow cytometry. Labeled antibodies can be detected using immunohistochemistry. Labeled antibodies can be detected using immunocytochemistry. Labeled antibodies can be detected using Western blotting.

[0462] sequence

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489] Example

[0490] These embodiments are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0491] Example 1: Design, expression, and analysis of recombinant antibody 2TdCH2-LFLEPS design

[0492] Materials and methods

[0493] Unless otherwise stated, all materials were purchased from Fisher Scientific (Waltham, MA).

[0494] Production of overexpression constructs of anti-OCT4-2TdCH2-LFLEPS, anti-P-perkinin-2TdCH2-LFLEPS, anti-IDO1-2TdCH2-LFLEPS, anti-FLAG-tag-2TdCH2-LFLEPS, anti-pERK1 / 2-2TdCH2, anti-PSD-95-2TdCH2, anti-TIGIT 2TdCH2-LFLEPS, and rabbit anti-mouse IgG-2TdCH2-LFLEPS antibodies.

[0495] Heavy chain expression construct: A backbone vector containing the rabbit 2TdCH2-LFLEPS antibody backbone was synthesized in pCDNA3.4, with annotated restriction sites as follows. Figure 2A As shown.

[0496] To generate expression constructs of OCT4 antibody and peracustrin antibody with a 2TdCH2-LFLEPS backbone, the Fab regions of parental OCT4 (clone ID: 3H8L6) and peracustrin antibody (clone ID: 21H24L9) were cloned into the restriction sites XBA1 and KAS1 in the aforementioned vector. Restriction analysis of the constructs confirmed the accuracy of the expression constructs. Figure 2C and Figure 3C ).

[0497] Light chain expression constructs: Light chain constructs of rabbit OCT4 (clone ID: 3H8L6) and perkinin (clone ID: 21H24L9) are presented in (… Figure 2B and Figure 3B As explained in Table 27 below. Similarly, other targets were cloned into the rabbit 2TdCH2-LFLEPS or mouse 2TdCH2 scaffold, as shown in Table 27 below.

[0498]

[0499] Expression and production of 2TdCH2-LFLEPS recombinant antibody

[0500] Using ExpiFectamine ™ 293 Transfection Kit (Product # A14524, Protocol Manual: MAN0007814) or ExpiFectamine ™ The CHO transfection kit (product # A29129, protocol manual: MAN0014337) transfects the heavy and light chains of the targets listed in Table 27 into Expi293F at a 1:1 ratio. ™ Cells. Five or eight days after transfection, the supernatant was collected and subjected to POROS. ™ MabCapture ™ A / POROS ™ MabCapture ™ G / CaptureSelect ™ IgG-Fc (multi-species) affinity matrix was purified by column chromatography and prepared in PBS pH 7.4. The antibody was then run through an HPLC column (BioSep). ™ 5µm SEC-s3000290Å, LC column 300×7.8mm, Ea) to check monomer content.

[0501] Immunological assay:

[0502] Immunocytochemistry: Immunofluorescence analysis was performed using 70% confluence of the desired cell line. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 2% BSA at room temperature for 1 h. Cells were stained with appropriate antibodies. Imaging was performed at 60X, 40X, or 20X magnification. ProLong was used. ™ Diamond anti-fading sealant was used to stain cell nuclei with DAPI (product # P36962) or Hoescht (product # H1399) and cytoskeleton F-actin staining was performed using rhodamine phalloidin (product # R415).

[0503] Western blotting: using Novex ® NuPAGE ® 4-12% Bis-Tris Gel (Product # NP0322BOX), XCellSureLock ™ Electrophoresis system (product # EI0002) and Novex ® Sharp pre-stained protein standards (product # LC5800) were used to perform electrophoresis on a known amount of protein sample. The eluted proteins were then analyzed using iBlot. ® The dried blot system (product #IB21001) transferred the material onto a nitrocellulose membrane and detected it using indicated primary and secondary antibodies. Novex was used. ® ECL Chemiluminescence Substrate Kit (Product # WP20005) or Chemiluminescence SuperSignal ™ The West Dura time-delay substrate (product #34075) was used for testing, and iBright was used for analysis. ™ FL 1500 (product # A44115) was tested.

[0504] Immunohistochemistry: To expose target proteins on tissue sections, eBioscience solutions diluted to 1X aqueous solution were used in a development chamber at 110°C. ™ IHC antigen retrieval solution - high pH (10X) (product # 00-4956-58) was used to perform heat-induced epitope retrieval on deparaffin sections for 15 minutes. After antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS at room temperature for 45 minutes, and then probed overnight in a humidified chamber at 4°C with or without an indicated primary antibody at a 1:100 dilution in 0.1% normal goat serum. The secondary antibody Alexa Fluor, which is highly cross-adsorbed with goat anti-rabbit IgG (H+L), was used.™ Plus 488 (product # A32731) was tested at 0.1% normal goat serum at a 1:2,000 dilution for 1 hour at room temperature. ReadyProbes was used. ™ The Tissue Autofluorescence Quenching Kit (Product # R37630) quenches autofluorescence from tissues. Cell nuclei are stained with DAPI (Product # D1306) and ProLong is used. ™ Glass anti-fading sealant (product #P36984) sealant for slices. (EVOS) ™ Images are captured at 20x magnification and externally deconvoluted on the M7000 imaging system (product # AMF7000).

[0505] Flow cytometry: Cells were fixed and permeabilized using either the intracellular fixation and permeabilization buffer kit (product # 88-8824-00) (for PerkinIndex) or the Foxp3 / transcription factor staining buffer kit (product # 00-5523-00) (for OCT4) and stained with the corresponding antibodies. Proteins of interest staining is represented by solid line histograms, and unstained populations are represented by dashed line histograms. Figure 10 It is a perkinin. Figures 12A-12B (For IDO1). Analysis was performed using live cells, such as by immobilizing the viability dye eFluor. ™ Determined by 450 (product # 65-0863-14) or the purple fluorescent reactive dye (L34964 A). Using Attune. ™ The NxT flow cytometer (product # A29004) acquires flow cytometry data.

[0506] Adaptation: For Alexa Fluor ™ Add 555 dye conjugation, and dialyze the antibody overnight at 4°C with bicarbonate buffer (0.05 M; pH 8.5). Mix 15 molar excess of dye with the antibody and incubate at room temperature in the dark for 1 hour. The sample is then analyzed by passing it through a Dowex filter. ™ Excess dye was removed using a 1×4-400 column with 50% slurry (product #22858). The antibody conjugate was formulated on StabilZyme containing EDTA. ® In the protein-free stabilizer (pH 6.3-7.3), the final concentration is 0.1 mM.

[0507] For Alexa Fluor ™Add 647 dye conjugation, and dialyze the antibody overnight at 4°C with bicarbonate buffer (0.05 M; pH 8.5). Mix 11 molar excess of dye with the antibody and incubate at room temperature in the dark for 1 hour. The sample is then analyzed by passing it through a Dowex filter. ™ Excess dye was removed using a 1×4-400 column with 50% slurry (product #22858). The antibody conjugate was formulated on StabilZyme containing EDTA. ® In the protein-free stabilizer (pH 6.3-7.3), the final concentration is 0.1 mM.

[0508] For Alexa Fluor ™ 488 dyes SiteClick ™ The conjugation process first modifies the carbohydrate domain of the antibody and then uses SiteClick. ™ Azide attachment was performed using an antibody azide modification kit (product # S10901). Then, SiteClick was used for antibody labeling of Alexa Fluor 647. ™ The sDIBO alkyne kit (product # S10906) labels sDIBO alkynes (Alexa Fluor) ™ 647-sDIBO dye is conjugated with azide-modified antibodies. The sample is then passed through Zeba... ™ Dye and biotin removal: Remove excess dye by rotating the column and filter plate 2 mL (product # A44299). Prepare the antibody conjugate in 1X Tris pH 7.0 (or TBS) with 0.02% sodium azide.

[0509] List of antibodies used for immunoassays: Unless otherwise stated, all antibodies were obtained from Thermo Fisher Scientific. Parental recombinant rabbit monoclonal antibody against Perkinin (21H24L9) (Product # 702785); Parental recombinant rabbit monoclonal antibody against OCT4 (3H8L6) (Product # 701756); Parental FLAG-tagged recombinant rabbit monoclonal antibody against FLAG (8H8L17) (Product # 701629); Parental recombinant rabbit monoclonal antibody against IDO1 (7H8L17) (Product # 702743); Parental recombinant mouse monoclonal antibody against TIGIT (MBSA43) (Product # 740010M); Parental recombinant mouse monoclonal antibody against PSD-95 (7E3-1B8) (Product # MA1-046); Goat anti-rabbit IgG (heavy chain), Superclonal ™ Recombinant secondary antibody, Alexa Fluor ™647 (Product # A27040); Goat anti-rabbit IgG (H+L) poly-HRP secondary antibody, HRP (Product # 32260); Goat anti-rabbit IgG (heavy chain), Superclonal ™ Recombinant secondary antibody, HRP (product # A27036); Goat anti-rabbit IgG (H+L) polyclonal secondary antibody, HRP (catalog number # 31460); Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor ™ Plus 488 (Product #A32790), donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor ™ Plus 488 (Product # A32795), Goat Anti-Mouse IgG (H+L) Superclonal ™ Secondary antibody (product # A28177).

[0510] List of reagents used for immunoassays: Unless otherwise specified, all reagents were obtained from Thermo Fisher Scientific. Flow cytometry staining buffer (product # 00-4222-26), IC fixation buffer (product # 00-8222-49), Foxp3 / transcription factor staining buffer kit (product # 00-5523-00), and immobilizable viable dye eFluor. ™ 450 (Product # 65-0863-14), a purple fluorescent reactive dye for 405nm excitation (Product # L34964 A), DAPI (Product # D1306), Hoescht 33342, trihydrochloride trihydrate (Product # H1399), ProLong ™ (Glass anti-fading sealant (product # P36984))

[0511] siRNA: OCT4-specific siRNA (Silencer) ® Select products #s10871, s10873), Perkinin-specific siRNA (Silencer) ® Select products # s530941 and s530942.

[0512] result

[0513] Production of rabbit and mouse recombinant antibodies with Fc engineered backbone

[0514] To evaluate the role of the 2TdCH2 Fc engineered backbone, three rabbit antibodies were generated, in which the heavy chain (HC) Fab region (SEQ ID NO: 8) and light chain (LC) Fab region (SEQ ID NO: 11) were derived from perkinin (clone ID: 21H24L9, product #702785), or the HC Fab region (SEQ ID NO: 33) and LC Fab region (SEQ ID NO: 35) were derived from OCT4 (clone ID: 3H8L6, product #701756), or the HC Fab region (SEQ ID NO: 78) and LC region (SEQ ID NO: 76) were derived from IDO1 (clone ID: 7H8L17, product #702743) rabbit recombinant antibody. The HC Fab region was cloned into a vector containing rabbit 2TdCH2 with or without the LFLEPS mutation (SEQ ID NO: 55). Antibodies were also generated in which these Fab clones were incorporated into vectors containing one additional Fc unit (1TdFc) of a nucleic acid having SEQ ID NO: 57 (encoding amino acid SEQ ID NO: 56) or two additional Fc units (2TdFc) of a nucleic acid having SEQ ID NO: 61 (encoding amino acid SEQ ID NO: 60). Figures 1A-1E Schematic diagrams of different engineering designs are provided. The yield, purity, and stability of these constructs are compared (Tables 28 and 29). Antibodies with the 2TdCH2 or 2TdCH2-LFLEPS backbone are equivalent to the parental wild-type antibodies in terms of yield, purity, and stability (Tables 28 and 29). Conversely, antibodies with the 1TdFc or 2TdFc backbone exhibit poorer yield, lower monomer purity, and stability compared to the parental wild-type antibodies (Tables 28 and 29).

[0515] To evaluate whether the engineered rabbit Fc backbone could confer benefits in chimeric antibodies containing rabbit Fc, a mouse-rabbit chimeric recombinant antibody against TIGIT was generated, wherein the HC Fab region (SEQ ID NO: 157) and LC region (SEQ ID NO: 155) were derived from a mouse TIGIT antibody (clone ID: MBSA43, product #16-9500-82) and cloned into a vector containing rabbit HC 2TdCH2 with or without the LFLEPS mutation (SEQ ID NO: 55). Figure 1EA schematic diagram of the engineering design is provided. To evaluate the effect of the 2TdCH2+LFLEPS Fc engineered backbone on rabbit secondary antibody, rabbit anti-mouse IgG (RAM) secondary antibody was generated, wherein the HC Fab region (SEQ ID NO: 138) was taken from rabbit anti-mouse secondary antibody (clone ID: RAM35HC35LC37) and cloned into a vector containing rabbit 2TdCH2 with or without LFLEPS mutation (SEQ ID NO: 53). Figure 1D and Figure 1E The diagrams for the engineering design are provided in the document.

[0516] In addition, the effect of 2TdCH2 Fc design on mouse antibodies was investigated. The mouse IgG1 Fc domain lacks the amino acid mediating the interaction with FcγR1, therefore the LFLEPS mutation does not confer the additional benefit of reduced Fc allotype background. Therefore, mouse antibodies were generated where the HC Fab region (SEQ ID NO: 97) was derived from mouse pERK1 / 2 (clone ID: MILAN8R, product #14-9109-82) or the HC Fab region (SEQ ID NO: 122) was derived from mouse PSD-95 hybridoma antibody (clone ID: 7E3-1B8, product #MA1-046) and cloned into a vector containing mouse IgG1 2TdCH2. Figure 1D The document provides schematic diagrams of the engineering design.

[0517]

[0518] Table 29: Summary of yield, purity, sensitivity and stability of engineered constructs of perkinin.

[0519]

[0520] Antibodies against Perkinin and OCT4 with the 2TdCH2 backbone exhibited increased sensitivity in Western blotting and immunocytochemistry.

[0521] First, the performance of the engineered 2TdCH2 antibody was compared with that of the parental antibody. In Western blots across different cell and / or tissue models, both the Parkinson's 2TdCH2 antibody and the OCT4 2TdCH2 antibody showed a significant increase in sensitivity, approximately twi...

Claims

1. An isolated polypeptide comprising the amino acid sequence of formula I: Z-(XY) n -Z r ) p -X, where Z stands for hinge; X is the CH2 domain; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer that is either 0 or 1.

2. The isolated polypeptide according to claim 1, wherein... Z contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102; X contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer that is either 0 or 1.

3. The isolated polypeptide according to claim 1 or claim 2, wherein Y comprises the amino acid sequence of GS, GGS having at least 90% identity with SEQ ID NO: 14, at least 90% identity with SEQ ID NO: 38, at least 90% identity with SEQ ID NO: 43, or at least 90% identity with SEQ ID NO:

44.

4. The isolated polypeptide according to any one of claims 1 to 3, wherein Z comprises SEQ ID NO: 18, SEQ ID NO: 48 or SEQ ID NO: 102; X comprises SEQ ID NO: 19, SEQ ID NO: 49 or SEQ ID NO: 104; Y comprises GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO: 44; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer of 0 or 1.

5. The isolated polypeptide according to any one of claims 1 to 4, wherein Z comprises the amino acid sequence of SEQ ID NO: 18, X comprises the amino acid sequence of SEQ ID NO: 19, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

6. The isolated polypeptide according to any one of claims 1 to 4, wherein Z comprises the amino acid sequence of SEQ ID NO: 48, X comprises the amino acid sequence of SEQ ID NO: 49, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

7. The isolated polypeptide according to any one of claims 1 to 4, wherein Z comprises the amino acid sequence of SEQ ID NO: 102, X comprises the amino acid sequence of SEQ ID NO: 104, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

8. The isolated polypeptide according to any one of the preceding claims, wherein the isolated polypeptide is linked to a second polypeptide via one or more disulfide bonds, wherein the isolated polypeptide and the second polypeptide share the same amino acid sequence.

9. The isolated polypeptide according to any one of the preceding claims, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO:

55.

10. The isolated polypeptide according to claim 8, wherein one or more disulfide bonds are present between the hinge of the isolated polypeptide and the hinge of the second polypeptide.

11. The isolated polypeptide according to any one of the preceding claims, wherein the isolated polypeptide further comprises a CH3 domain.

12. The isolated polypeptide according to claim 9, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

106.

13. An antibody comprising a separated polypeptide according to any one of the preceding claims linked to a Fab domain.

14. The antibody of claim 13, wherein the Fab comprises a CH1 domain.

15. The antibody according to claim 14, wherein the CH1 domain comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 100 or SEQ ID NO:

160.

16. The antibody of claim 13, wherein the Fab comprises a Fab heavy chain and a Fab light chain paired to form a binding domain.

17. The antibody according to any one of claims 13 to 16, wherein the Fab is immune-specifically bound to perkinin, OCT4, IDO1, FLAG tag, pERK1 / 2, PSD-95 or TIGIT.

18. The antibody according to any one of claims 13 to 17, wherein the isolated polypeptide is an antibody having isotype IgG.

19. An antibody comprising a polypeptide containing the amino acid sequence of Formula I: Z-(XY) n -Z r ) p -X, where Z stands for hinge; X is the CH2 domain; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer that is either 0 or 1.

20. The antibody according to claim 19, wherein Z contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102; X contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer that is either 0 or 1.

21. The antibody according to claim 19 or claim 20, wherein Y comprises an amino acid sequence of GS, GGS having at least 90% identity with SEQ ID NO: 14, at least 90% identity with SEQ ID NO: 38, at least 90% identity with SEQ ID NO: 43, or at least 90% identity with SEQ ID NO:

44.

22. The antibody according to any one of claims 19 to 21, wherein Z comprises SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102; X comprises SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104; Y comprises GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer of 0 or 1.

23. The antibody according to any one of claims 19 to 22, wherein Z comprises the amino acid sequence of SEQ ID NO: 18, X comprises the amino acid sequence of SEQ ID NO: 19, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

24. The antibody according to any one of claims 19 to 22, wherein Z comprises the amino acid sequence of SEQ ID NO: 48, X comprises the amino acid sequence of SEQ ID NO: 49, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

25. The antibody according to any one of claims 19 to 22, wherein Z comprises the amino acid sequence of SEQ ID NO: 102, X comprises the amino acid sequence of SEQ ID NO: 104, Y comprises the amino acid sequence of SEQ ID NO: 44, n is 1, and p is 2.

26. The antibody according to any one of claims 19 to 25, wherein the polypeptide is linked to the second polypeptide via one or more disulfide bonds, wherein the polypeptide and the second polypeptide share the same amino acid sequence.

27. The antibody according to any one of claims 19 to 26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO:

55.

28. The antibody of claim 26, wherein one or more disulfide bonds are present between the hinge of the polypeptide and the hinge of the second polypeptide.

29. The antibody according to any one of claims 19 to 28, wherein the antibody further comprises a CH3 domain.

30. The antibody according to claim 29, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

106.

31. The antibody according to any one of claims 13 to 30, wherein the background signal of the antibody is reduced by at least 1 / 3 compared to the corresponding wild-type antibody that does not contain the isolated polypeptide.

32. The antibody according to any one of claims 13 to 31, wherein the sensitivity of the antibody is increased by at least 0.5 times compared to the corresponding wild-type antibody that does not contain the isolated polypeptide.

33. The isolated polypeptide according to any one of claims 1-12 or the antibody according to any one of claims 13-32, wherein the isolated polypeptide or the antibody further comprises a label.

34. A nucleic acid encoding a polypeptide isolated according to any one of claims 1-12 or an antibody according to any one of claims 13-32.

35. An antibody that specifically binds to perkinin, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 13, an LCDR2 comprising the amino acid sequence RAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

36. The antibody according to claim 35, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

37. The antibody according to claim 35 or claim 36, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

48.

38. The antibody according to any one of claims 35 to 37, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

39. The antibody according to any one of claims 35 to 38, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

40. The antibody according to any one of claims 35 to 39, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

16.

41. The antibody according to any one of claims 35 to 40, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 3 and a light chain having the polypeptide sequence of SEQ ID NO:

12.

42. The antibody according to any one of claims 35 to 41, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 2 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

11.

43. An antibody that specifically binds to OCT4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 37, an LCDR2 comprising the amino acid sequence GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 39, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

44. The antibody according to claim 43, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

45. The antibody according to claim 43 or claim 44, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

48.

46. ​​The antibody according to any one of claims 43 to 45, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

47. The antibody according to any one of claims 43 to 46, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

48. The antibody according to any one of claims 43 to 47, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 32 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

40.

49. The antibody according to any one of claims 43 to 48, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 28 and a light chain having the polypeptide sequence of SEQ ID NO:

36.

50. The antibody according to any one of claims 43 to 49, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 27 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

35.

51. An antibody that specifically binds to IDO1, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 66, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 68, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 73, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

52. The antibody according to claim 51, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

53. The antibody according to claim 51 or claim 52, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

48.

54. The antibody according to any one of claims 51 to 53, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

55. The antibody according to any one of claims 51 to 54, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

56. The antibody according to any one of claims 51 to 55, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 65 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

71.

57. The antibody according to any one of claims 51 to 56, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 75 and a light chain having the polypeptide sequence of SEQ ID NO:

77.

58. The antibody according to any one of claims 51 to 57, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 74 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

76.

59. An antibody that specifically binds to a FLAG tag, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 163, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 164, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 168, an LCDR2 comprising the amino acid sequence of YAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 169, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

60. The antibody according to claim 59, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

61. The antibody according to claim 59 or claim 60, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

48.

62. The antibody according to any one of claims 59 to 61, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

63. The antibody according to any one of claims 59 to 62, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

64. The antibody according to any one of claims 59 to 63, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

167.

65. The antibody according to any one of claims 59 to 64, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 171 and a light chain having the polypeptide sequence of SEQ ID NO:

173.

66. The antibody according to any one of claims 59 to 65, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 170 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

172.

67. An antibody as a secondary antibody against rabbit anti-mouse IgG, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 132, an LCDR2 comprising the GAS amino acid sequence, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 133, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

68. The antibody according to claim 67, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

69. The antibody according to claim 67 or claim 68, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

48.

70. The antibody according to any one of claims 67 to 69, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

71. The antibody according to any one of claims 67 to 70, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

72. The antibody according to any one of claims 67 to 71, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

131.

73. The antibody according to any one of claims 67 to 72, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 135 and a light chain having the polypeptide sequence of SEQ ID NO:

137.

74. The antibody according to any one of claims 67 to 73, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 134 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

136.

75. An antibody that specifically binds to pERK1 / 2, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 85, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 87, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 91, an LCDR2 comprising the amino acid sequence of KVS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

76. The antibody according to claim 75, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO:

104.

77. The antibody according to claim 75 or claim 76, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO:

102.

78. The antibody according to any one of claims 75 to 77, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

79. The antibody according to any one of claims 75 to 78, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

106.

80. The antibody according to any one of claims 75 to 79, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 83 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

90.

81. The antibody according to any one of claims 75 to 80, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 94 and a light chain having the polypeptide sequence of SEQ ID NO:

96.

82. The antibody according to any one of claims 75 to 81, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 93 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

95.

83. An antibody that specifically binds to PSD-95, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 111, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 112, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 113, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 116, an LCDR2 comprising the amino acid sequence of DTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 117, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

84. The antibody according to claim 83, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO:

104.

85. The antibody according to claim 83 or claim 84, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO:

102.

86. The antibody according to any one of claims 83 to 85, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

87. The antibody according to any one of claims 83 to 86, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

106.

88. The antibody according to any one of claims 83 to 87, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

115.

89. The antibody according to any one of claims 83 to 88, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 119 and a light chain having the polypeptide sequence of SEQ ID NO:

121.

90. The antibody according to any one of claims 83 to 89, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 118 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

120.

91. An antibody that specifically binds to TIGIT, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) paired to form a binding domain, the binding domain comprising: a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 148, a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 151, an LCDR2 comprising the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 152, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are connected by a first linker and optionally a first hinge, and wherein the second CH2 domain and the third CH2 domain are connected by a second linker and optionally a second hinge, and wherein the heavy chain further comprises a CH3 domain.

92. The antibody according to claim 91, wherein the first CH2 domain, the second CH2 domain and the third CH2 domain each comprise the amino acid sequence of SEQ ID NO:

19.

93. The antibody according to claim 91 or claim 92, wherein the first hinge and the second hinge each comprise the amino acid sequence of SEQ ID NO:

18.

94. The antibody according to any one of claims 91 to 93, wherein the first adapter and the second adapter each comprise the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43 or SEQ ID NO:

44.

95. The antibody according to any one of claims 91 to 94, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO:

20.

96. The antibody according to any one of claims 91 to 95, wherein the antibody comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 145 and a light chain variable region having a polypeptide sequence of SEQ ID NO:

150.

97. The antibody according to any one of claims 91 to 96, wherein the antibody comprises a heavy chain having the polypeptide sequence of SEQ ID NO: 154 and a light chain having the polypeptide sequence of SEQ ID NO:

156.

98. The antibody according to any one of claims 91 to 97, wherein the antibody comprises a heavy chain encoded by the polynucleotide sequence of SEQ ID NO: 153 and a light chain encoded by the polynucleotide sequence of SEQ ID NO:

155.

99. The antibody according to any one of claims 13 to 32 or 35 to 98, wherein the antibody provides at least a 0.5-fold increase in signal enhancement compared to the corresponding wild-type antibody.

100. The antibody according to any one of claims 13 to 33 or 35 to 99, wherein the antibody provides at least a 1 / 3 reduction in background signal compared to the corresponding wild-type antibody.

101. A method for detecting the presence of a target protein in a sample, the method comprising: The isolated polypeptide according to any one of claims 1 to 12 or the antibody according to any one of claims 13 to 33 or 35 to 100 is applied to a sample containing the target protein. Under conditions that allow the isolated polypeptide or the antibody to bind to the target protein and form bound isolated polypeptide or bound antibody, the isolated polypeptide or the antibody in the sample is incubated. The presence of the bound isolated polypeptide or the bound antibody is determined to detect the presence of the target protein.

102. The method of claim 101, wherein the isolated polypeptide or antibody has less background binding to cells expressing the Fcγ receptor compared to the wild-type isolated polypeptide or wild-type antibody.

103. The method according to claim 101 or claim 102, wherein the target protein is OCT4, perkinin, IDO1, FLAG tag, pERK1 / 2, PSD-95, or TIGIT.

104. The method according to any one of claims 101 to 103, wherein the target protein is present in the sample in low abundance.

105. The method according to any one of claims 101 to 104, wherein the presence of the bound isolated polypeptide or the bound antibody is determined by applying a secondary antibody to the sample, wherein the secondary antibody is labeled.

106. The method according to any one of claims 101 to 105, wherein the antibody is labeled.

107. The method of claim 105 or claim 106, wherein the label is selected from fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, polymer dyes, particles, electron transfer agents, haptens, enzymes, radioisotopes, chromophores, and oligonucleotide-based fluorescent dyes.

108. The method according to claim 107, wherein the fluorophore is pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzoindole, oxazole or benzoxazole, thiazole or benzothiazole, indene, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), anthocyanin, carbonyl anthocyanin, benzoanthocyanin, quinolone, porphyrin, salicylate, anthranilate, azulene, perylene, pyridine, quinoline, boron polyacrylamide, xanthannaphthalene, rhodamine, silyl rhodamine, fluorescein, p-methylaminophenol, rhosamine, oxazine or benzoxazine, benzophenoxazine, carbazine, finarenone, coumarin, benzofuran, benzeninone, benzopyranone, styraxene, squaricine, halogen, anthraquinone or quinazolinone or conjugated polymer dye.

109. The method of claim 107, wherein the particles are semiconductor nanocrystals.

110. The method of claim 107, wherein the hapten is biotin or a biotin derivative.

111. The method of claim 107, wherein the enzyme is selected from peroxidase, phosphatase, glycosidase and luciferase.

112. The method of claim 111, wherein the method further comprises adding an enzyme substrate selected from chromogenic substrates, fluorescent substrates, and chemiluminescent substrates.

113. The method according to any one of claims 106 to 112, wherein the detection of the labeled antibody is performed using flow cytometry, immunohistochemistry, immunocytochemistry, or Western blotting.

114. The method according to any one of claims 101 to 113, wherein the isolated polypeptide is an antibody having isotype IgG.

115. A method for increasing the sensitivity of an antibody, the method comprising: Introducing a polypeptide comprising the amino acid sequence of formula I into the heavy chain of the antibody: Z-(XY) n -Z r ) p -X, where Z stands for hinge; X is the CH2 domain; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer of 0 or 1. The sensitivity of the antibody is increased by at least 0.5 times compared to the corresponding wild-type antibody.

116. The method of claim 115, further comprising introducing an LFLEPS mutation into the amino acid sequence of X, wherein the LFLEPS mutation comprises L234F, L235E, and P331S substitutions.

117. The method according to claim 115 or claim 116, wherein X comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 19, at least 90% identity with SEQ ID NO: 49, or at least 90% identity with SEQ ID NO:

104.

118. The method according to any one of claims 115 to 117, wherein Z comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 18, at least 90% identity with SEQ ID NO: 48, or at least 90% identity with SEQ ID NO:

102.

119. The method according to any one of claims 115 to 118, wherein Y comprises an amino acid sequence having at least 90% identity with the amino acid sequence of GS, GGS, SEQ ID NO:14, SEQ ID NO:38, SEQ ID NO:43 or SEQ ID NO:

44.

120. A method for reducing the background signal of an antibody, the method comprising: Introducing a polypeptide comprising the amino acid sequence of formula I into the heavy chain of the antibody: Z-(XY) n -Z r ) p -X, where Z stands for hinge; X is the CH2 domain; Y stands for connector; n is an integer of 1 or greater; p is an integer of 2 or greater; and r is an integer that is either 0 or 1; The background signal of the antibody was reduced by at least one-third compared to the corresponding wild-type antibody.

121. The method of claim 120, further comprising introducing an LFLEPS mutation into the amino acid sequence of X, wherein the LFLEPS mutation comprises L234F, L235E, and P331S substitutions.

122. The method according to claim 120 or claim 121, wherein X comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 19, at least 90% identity with SEQ ID NO: 49, or at least 90% identity with SEQ ID NO:

104.

123. The method according to any one of claims 120 to 122, wherein Z comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 18, at least 90% identity with SEQ ID NO: 48, or at least 90% identity with SEQ ID NO:

102.

124. The method according to any one of claims 120 to 123, wherein Y comprises an amino acid sequence having at least 90% identity with the amino acid sequence of GS, GGS, SEQ ID NO:14, SEQ ID NO:38, SEQ ID NO:43 or SEQ ID NO:44.

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