Manipulated antibodies and their use

Abstract

This specification discloses isolated polypeptides and antibodies having improved sensitivity and reduced background signal, as well as methods for using them. Specifically, the antibody comprises a polypeptide containing a sequence-defined CH2 domain linked to a hinge domain.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefits under Section 119(e) of the U.S. Patent Act for Provisional Indian Application No. IN202441066590, filed on 3 September 2024, Provisional Indian Application No. IN202341061917, filed on 14 September 2023, and Provisional Indian Application No. IN202341061835, filed on 14 September 2023. The entire contents of the aforementioned applications are incorporated herein by reference.

[0002] Reference to electronic sequence listings This application includes a sequence listing submitted electronically with this specification in .XML format, which is incorporated herein by reference in its entirety. The .XML copy, created on September 10, 2024, is named "TP385350WO1.xml" and has a size of 279,164 bytes. The sequence listing contained in this .XML file is part of this specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to isolated polypeptides and antibodies having improved sensitivity, and to methods for using them. [Background technology]

[0004] Recombinant antibodies offer the ability to rationally manipulate beneficial properties. One such beneficial property is antibody sensitivity, i.e., the intensity of the signal produced in a particular immunoassay. Highly sensitive antibodies have several advantages, such as the ability to detect low-abundance or rare targets and target proteins in challenging imaging assays. Antibody sensitivity can be increased by affinity maturation of the antibody clone, involving mutations in the antigen-binding site or paratope. However, affinity maturation must be adjusted for each antibody under consideration and is therefore not a scalable solution for dealing with a large number of antibodies. In addition, introducing mutations into the paratope alters the antibody clone and essentially produces a new antibody. For example, improved approaches are needed to increase antibody sensitivity by rational modification of the antibody Fc region. The disclosed peptides, antibodies, compositions, and their uses address these and other important needs. [Overview of the project]

[0005] In this specification, formula I:Z-(XY n -Z r ) p An isolated polypeptide comprising the amino acid sequence -X is disclosed, where Z is a hinge region, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1. In certain embodiments, the isolated polypeptide has at least a 1.5-fold increase in signal enhancement or sensitivity. In certain embodiments, the isolated polypeptide has at least a 1.5-fold reduction in background signal.

[0006] In this specification, the formula I:Z-(XY n -Z r ) pAn antibody is disclosed comprising a polypeptide containing the amino acid sequence -X, where Z is a hinge region, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1. In certain embodiments, the antibody has at least a 1.5-fold increase in signal enhancement or sensitivity compared to the corresponding wild-type antibody. In certain embodiments, the antibody has at least a 1.5-fold reduction in background signal compared to the corresponding wild-type antibody.

[0007] This specification discloses an antibody that binds immunospecifically to parkin, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 5, HCDR3 containing the amino acid sequence of SEQ ID NO: 6, and a light chain complementarity determining region 1 containing the amino acid sequence of SEQ ID NO: 13. A binding domain is formed comprising 1. LCDR1), LCDR2 containing the amino acid sequence RAS, and LCDR3 containing the amino acid sequence of Sequence ID No. 15, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0008] This specification discloses an antibody that immunospecifically binds to OCT4, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 31, a light chain complementarity determination 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0009] This specification discloses an antibody that immunospecifically binds to IDO1, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 68, 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0010] This specification discloses an antibody that immunospecifically binds to a FLAG-tag, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising: a heavy chain complementarity determination 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 165; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, the second and third CH2 domains being linked by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0011] In this specification, rabbit anti-mouse (rabbit An anti-mouse (RAM) secondary antibody is disclosed, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising a heavy chain complementarity determination region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 127, HCDR2 comprising the amino acid sequence of SEQ ID NO: 128, HCDR3 comprising the amino acid sequence of SEQ ID NO: 129, a light chain complementarity determination region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 132, LCDR2 comprising the amino acid sequence of GAS, and 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, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0012] This specification discloses an antibody that immunospecifically binds to pERK1 / 2, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 85, HCDR2 containing the amino acid sequence of SEQ ID NO: 86, HCDR3 containing the amino acid sequence of SEQ ID NO: 87, a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, LCDR2 containing the amino acid sequence of KVS, and LCDR3 containing 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, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0013] This specification discloses an antibody that immunospecifically binds to PSD-95, the antibody comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising a heavy chain complementarity determination 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 determination 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0014] Disclosed herein is an antibody that immunospecifically binds to TIGIT, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair 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, 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 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, the second CH2 domain and the third CH2 domain are connected by a second linker and an optional second hinge, and the heavy chain further comprises a CH3 domain.

[0015] Disclosed herein is a method of detecting the presence of a target protein in a sample, the method comprising applying an isolated polypeptide disclosed herein or an antibody disclosed herein to the sample containing the target protein, incubating the isolated polypeptide or the antibody in the sample under conditions that allow the isolated polypeptide or the polypeptide or the antibody to bind to the target protein to form a bound isolated polypeptide or a bound polypeptide or a bound antibody, and determining the presence of the bound isolated polypeptide or the bound polypeptide or the bound antibody, thereby detecting the presence of the target protein.

[0016] Disclosed herein is a method of increasing the sensitivity of an antibody, wherein the heavy chain of the antibody is modified with Formula I: Z-(X-Y n -Z r ) p-The method comprises introducing a polypeptide containing the amino acid sequence of X, where Z is a hinge, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1, wherein the sensitivity of the antibody is increased by at least 1.5 times compared to the corresponding wild-type antibody. In certain embodiments, the method further comprises introducing an LFLEPS mutation into the amino acid sequence of X, where the LFLEPS mutation includes L234F, L235E, and P331S substitutions.

[0017] This specification provides a method for reducing the background signal of an antibody, wherein the heavy chain of the antibody is given the formula I:Z-(XY n -Z r ) p The method comprises introducing a polypeptide containing the amino acid sequence of -X, where Z is a hinge, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1, wherein the background signal of the antibody is reduced by at least 1.5 times compared to the corresponding wild-type antibody. In certain embodiments, the method further comprises introducing an LFLEPS mutation into the amino acid sequence of X, where the LFLEPS mutation includes L234F, L235E, and P331S substitutions. The above summary, as well as 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 specific embodiments shown in the drawings. [Brief explanation of the drawing]

[0018] [Figure 1A] This is a schematic diagram of a parental wild-type antibody (WT IgG, approximately 150 kDa). [Figure 1B] This is a schematic diagram of a 1TdFc design (IgG-2Fc, approximately 200 kDa). The 1TdFc design has one additional Fc domain connected by a linker and hinge. [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 linker and hinge. [Figure 1D] This is a schematic diagram of a 2TdCH2 design (approximately 200kDa). The 2TdCH2 design has two additional CH2 domains connected by a linker and an optional hinge. [Figure 1E] This is a schematic diagram of the 2TdCH2-LFLEPS design (approximately 200 kDa). The 2TdCH2-LFLEPS design has two additional CH2 domains connected by a linker and an optional hinge, as well as an FcR mutation (indicated by an asterisk) represented by the acronym "LFLEPS". [Figure 2A] This plasmid map shows the synthesis of a polynucleotide sequence of an anti-OCT4 antibody heavy chain (HC) with the 2TdCH2-LFLEPS configuration and its cloning 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. [Figure 2B] This plasmid map shows the synthesis of a polynucleotide sequence of the anti-OCT4 antibody light chain (LC) and its cloning between the XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-NotI restriction enzyme can be used to subclone another target LC "Fab" region. [Figure 2C] Restriction digestion analyses of anti-OCT4 antibody HC and anti-OCT4 antibody LC having the 2TdCH2-LFLEPS configuration are shown. Anti-OCT4 antibody HC 2TdCH2-LFLEPS shows a 2203 bp insert released upon XbaI-NotI digestion. Anti-OCT4 antibody LC shows a 746 bp insert released upon XbaI-NotI digestion. [Figure 3A]This plasmid map shows the synthesis of a polynucleotide sequence of an anti-Parkin antibody heavy chain (HC) with the 2TdCH2-LFLEPS configuration and its cloning 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. [Figure 3B] This plasmid map shows the synthesis of a polynucleotide sequence of an anti-Parkin antibody light chain (LC) and its cloning between the XbaI-NotI restriction sites in the pcDNA3.4 vector. The XbaI-NotI restriction enzyme can be used to subclone another target LC "Fab" region. [Figure 3C] Restriction digestion analyses of anti-parkin antibody HC and anti-parkin antibody LC having the 2TdCH2-LFLEPS configuration are shown. Anti-parkin antibody HC 2TdCH2-LFLEPS shows a 2218 bp insert released upon XbaI-NotI digestion. Anti-parkin antibody LC shows a 743 bp insert released upon XbaI-NotI digestion. [Figure 4A] This shows a typical HPLC profile of a parental anti-parkin wild-type (WT) antibody with 95% monomer purity. [Figure 4B] This shows a typical HPLC profile of an anti-OCT4 1TdFc antibody with a monomer purity of 63%. [Figure 4C] A typical HPLC profile of an anti-Parkin 2TdFc antibody with a monomer purity of 33% is shown. [Figure 4D] A typical HPLC profile of the anti-Parkin 2TdCH2-LFLEPS antibody with 87% monomer purity is shown. [Figure 5A] This is a Western blot (WB) analysis performed on whole cell extracts of rat brain tissue (left panel) and PC-12 cells (right panel). The blots were searched for wild-type (WT) and engineered anti-parkin antibodies (1TdFC, 2TdFc, 2TdCH2). A band of approximately 48 kDa corresponding to parkin was observed in the lysates of the tested rat brain and PC-12 cells. [Figure 5B] The Western blot (upper panel) shows how antibody specificity was demonstrated by siRNA-mediated knockdown of the target protein. SH-SY5Y cells were transfected with parkin siRNA, and a decrease in signal intensity was observed when applying a Western blot using the anti-parkin 2TdCH2-LFLEPS antibody. The lower panel is a graphical representation of the Western blot analysis. [Figure 5C] This is a Western blot analysis performed on whole cell extracts of PC-12 cells. The blots were searched for wild-type (WT) and engineered anti-parkin antibodies (2TdCH2, 2TdCH2-LFLEPS). Similar intensities of the approximately 48 kDa band corresponding to parkin were observed in PC-12 cell lysates, using 2TdCH2 and 2TdCH2-LFLEPS for comparison. [Figure 5D] This is a scatter plot analysis of WB data, showing that the anti-Parkin 2TdCH2 antibody exhibits a significant enhancement (**p<0.01) compared to the WT antibody. [Figure 6A] This is a Western blot analysis performed on whole cell extracts of NTERA-2 cells (left panel) and F9 cells (right panel). The blots were explored with wild-type (WT) and engineered anti-OCT4 antibodies (1TdFC, 2TdFc, 2TdCH2). A band of approximately 38 kDa corresponding to OCT4 was observed in the tested cell lysates. [Figure 6B] This was a Western blot analysis performed on whole cell extracts of NTERA-2 cells. The blots were searched for wild-type (WT) and engineered anti-OCT4 antibodies (2TdCH2, 2TdCH2-LFLEPS). Similar intensities of a band of approximately 38 kDa corresponding to OCT4 were observed in NTERA-2 cell lysates using anti-OCT4 2TdCH2 antibody and 2TdCH2-LFLEPS antibody for comparison. [Figure 6C]The antibody specificity was demonstrated by relative expression using BOI in NTERA-2, NCCIT, and F9 cells (upper panel). HEK293, A431, and HELA were negative cell lines. siRNA-mediated knockdown of the target protein (lower panel). NTERA-2 cells were transfected with OCT4 siRNA, and a decrease in signal intensity was observed by Western blotting using anti-OCT4 2TdCH2-LFLEPS antibody. [Figure 6D] This is a scatter plot analysis of WB data, showing that the anti-OCT4 2TdCH2 antibody exhibits a significant enhancement (****p<0.0001) compared to the WT antibody. [Figure 7A] This is a bar graph analysis of Western blot data obtained from anti-Parkin 2TdCH2 antibody using various secondary antibodies. The anti-Parkin 2TdCH2 antibody showed fulcrum enhancement across different secondary antibodies compared to the wild-type (WT) antibody. [Figure 7B] This is a bar graph analysis of Western blot data obtained from anti-OCT4 2TdCH2 antibody using various secondary antibodies. The anti-OCT4 2TdCH2 antibody showed fulcrum enhancement across different secondary antibodies compared to the wild-type (WT) antibody. [Figure 8A] This is an immunofluorescence analysis of OCT4 performed using 70% densely packed NTERA-2 cells. Cells were stained with different concentrations of anti-OCT4 wild-type (WT) antibody (upper panel) or anti-OCT4 2TdCH2-LFLEPS antibody (1:1,000) (lower panel). [Figure 8B] This study demonstrates that OCT4 knockdown was achieved by transfecting NTERA-2 cells with specific validated siRNAs (Silencer® select product numbers 10871 and s10873). Immunofluorescence analysis was performed on NTERA-2 cells transfected with OCT4-specific siRNAs (panels i-l) or non-specific scrambled siRNAs (panels e-h) (untransfected cells, panels a-d). [Figure 9A]This is an immunohistochemical analysis of Parkin performed on formalin-fixed, paraffin-embedded mouse kidney tissue sections. The sections were detected with anti-Parkin wild-type (WT) (lower panel) or anti-Parkin 2TdCH2-LFLEPS (upper panel) (1:100 dilution) antibody. [Figure 9B] This study involved immunohistochemical analysis of Parkin using formalin-fixed, paraffin-embedded mouse kidney tissue sections. The sections were detected with anti-Parkin polyclonal antibody (benchmark (BM) or positive control) (lower panel) or Parkin 2TdCH2-LFLEPS antibody (upper panel) (1:100 dilution). [Figure 10] This is a flow cytometry analysis of parkin performed in PC-12 cells. Cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (product number 88-8824-00), and then intracellularly stained with 2 μg of anti-parkin wild-type (WT) antibody, anti-parkin 2TdCH2-LFLEPS antibody, or positive control (PC) antibody (PA5-1339). Unstained samples were used as controls. The upper panel shows the flow cytometry spectrum, and the lower panel shows a graph of the flow data. [Figure 11] This graph shows the immunofluorescence analysis of IDO1 performed using 70% densely packed CD64-GFP(FcγR1) transfected HEK-293E cells, demonstrating the amount of FcγR1 binding by the antibody. Cells were stained with IDO1 wild-type (WT) ± LFLEPS antibody or anti-IDO1 2TdCH2 ± LFLEPS antibody at 0.5 μg / ml and 2.5 μg / ml, followed by staining with goat anti-rabbit Alexa Fluor® 647 secondary antibody (product number A21244). [Figure 12A]This is a flow cytometry analysis of IDO1 performed using 70% densely packed CD64-GFP(FcγR1) transfected HEK-293E cells. Cells were stained with anti-IDO1 wild-type ±LFLEPS(WT) antibody or anti-IDO1 2TdCH2 ±LFLEPS antibody at 0.5 μg / ml (lower panel) and 2.5 μg / ml (upper panel), followed by staining with goat anti-rabbit Alexa Fluor® 647 secondary antibody (product number A21244). Viable cells were used for analysis, and data were acquired using an Attune® NxT Flow Cytometer (product number A29004). Left shifts in the histogram represent background signal reduction due to LFLEPS mutations. [Figure 12B] Figure 12A shows a graph of the flow cytometry analysis, demonstrating the amount of FcγR1 binding by the antibody. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-tailed Student's T-test. [Figure 13A] This graph shows the results of immunofluorescence analysis of IDO1 performed using 70% densely packed CD64-GFP (FcγR1) transfected HEK-293E cells, with and without interferon-gamma (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 staining with goat anti-rabbit Alexa Fluor® 647 secondary antibody (product number A21244). [Figure 13B] This graph shows the results of immunofluorescence analysis of IDO1 performed using 70% densely packed CD64-GFP(FcγR1) transfected HEK-293E cells, 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 staining with goat anti-rabbit Alexa Fluor® 647 secondary antibody (product number A21244). [Figure 14A]This flow cytometry analysis demonstrates that the 2TdCH2-LFLEPS design in mouse-rabbit chimeric antibodies (Fab-Fc chimeras) enhances TIGIT signal detection in human PBMCs compared to the wild-type design. Human PBMCs were stained with CD4 FITC (product no. 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® Plus 647 secondary antibody (product no. A55060). Viable cells in lymphocyte gating were used for analysis, and data were acquired using an Attune® NxT Flow Cytometer (product no. A29004). [Figure 14B] Figure 14A shows a graph of flow cytometry analysis, illustrating the staining indices of various antibodies. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-tailed Student's t-test, and an asterisk (**) indicates statistical significance determined by p<0.01. [Figure 15] This graph shows the immunofluorescence analysis effect of the 2TdCH2 LFLEPS design on rabbit anti-mouse (RAM) IgG secondary antibody, illustrating the staining indices of various antibodies. Analysis was performed using 70% densely packed CD64-GFP(FcγR1) transfected HEK-293E cells. Cells were stained with 0.5 μg / ml and 2.5 μg / ml RAM wild-type (WT) antibody ± LFLEPS or RAM 2TdCH2-LFLEPS ± LFLEPS antibody, followed by staining with donkey anti-rabbit Alexa Fluor® 647 secondary antibody d(A32795) to detect RAM antibodies. Increased staining with RAM WT antibody and RAM 2TdCH2 antibody (without LFLEPS) indicates nonspecific binding of the secondary antibody, as the primary antibody was not present in the sample. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-sided Student's T-test. [Figure 16A]The left panel shows Western blots performed by loading whole cell extracts of jarcut cells with and without PMA treatment (200 ng / ml) using 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® secondary antibody (product number A28177). The right panel shows a graphical representation of the Western blot analysis. [Figure 16B] This is an immunofluorescence analysis performed on serum-deficient A431 cells treated with EGF (200 ng / ml) using 2.5 μg / ml anti-pERK1 / 2 WT antibody and anti-pERK1 / 2 2TdCH2 recombinant mouse monoclonal antibody, followed by goat anti-mouse IgG(H+L) Alexa Fluor® Plus 488 secondary antibody. The left panel shows the immunofluorescence images, and the right panel shows the immunofluorescence analysis graph. [Figure 17] Western blots are shown, performed by loading whole cell extracts of Neuro2A with anti-PSD-95 WT antibody and anti-PSD-95 2TdCH2 antibody at concentrations of 4.0-0.5 μg / ml, followed by goat anti-mouse IgG(H+L)HRP Superclonal® secondary antibody (product number A28177). [Figure 18A] This is an immunofluorescence analysis performed in NTERA2 cells using a 1 μg / ml wild-type or 2TdCH2 LFLEPS anti-OCT4-Alexa Fluor® Plus 555 antibody conjugate. [Figure 18B] Figure 18A shows a graphical representation of the immunofluorescence analysis. An asterisk (**) indicates statistical significance determined by p<0.01 using a two-tailed Student's t-test. [Figure 19A] This is an immunofluorescence analysis performed in NTERA2 cells using 1 μg / ml wild-type or 2TdCH2 LFLEPS anti-OCT4-Alexa Fluor® Plus 647 conjugate. [Figure 19B] Figure 19A shows a graphical representation of the immunofluorescence analysis. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-tailed Student's t-test. [Figure 20A] This is a Western blot analysis performed on whole cell lysates (WCL) of PC-12 cells using wild-type or 2TdCH2 LFLEPS anti-parkin HRP conjugate at 1 μg / ml in 4 and 8 molar ratios. [Figure 20B] This is a Western blot analysis performed on EXPI293 cells transfected with the FLAG-histone H4 construct, using wild-type or 2TdCH2 LFLEPS anti-FLAG-tagged HRP conjugate at 1 μg / ml in 4 and 8 molar ratios. [Figure 20C] This is a Western blot analysis performed on EXPI293 cells transfected with the FLAG-histone H4 construct, using wild-type or 2TdCH2 LFLEPS anti-FLAG-tagbiotin conjugate at 1 μg / ml in molar ratios of 25 and 50. [Figure 21A] This shows the degree of labeling (DOL) from SiteClick® conjugations of Alexa Fluor® 488 dye with anti-Parkin WT, 2TdCH2, and 2TdCH2 LFLEPS antibodies. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-tailed Student's t-test, and an asterisk (**) indicates statistical significance determined by p<0.01. [Figure 21B] This shows the degree of labeling (DOL) from SiteClick® conjugations of Alexa Fluor® 488 dye with anti-OCT4 WT, 2TdCH2, and 2TdCH2 LFLEPS antibodies. An asterisk (*) indicates statistical significance determined by p<0.05 using a two-tailed Student's T-test. [Modes for carrying out the invention]

[0019] Various publications, articles, and patents are cited or referenced in the background art and throughout this specification, and each of these references is incorporated in whole by reference. The considerations of documents, acts, materials, apparatus, articles, etc. included herein are for the purpose of providing context to the invention. Such considerations do not constitute an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0020] The polypeptides, antibodies, and methods disclosed may be more readily understood by referring to the following detailed description together with the accompanying drawings that form part of this disclosure. It should be understood that the polypeptides, antibodies, and methods disclosed are not limited to the specific polypeptides, antibodies, and methods described and / or illustrated herein, and that the terms used herein are for illustrative purposes only and are intended to describe specific embodiments and are not intended to limit the claimed polypeptides, antibodies, and methods.

[0021] Unless otherwise specifically stated, any description of possible mechanisms or modes of action or reasons for improvement is illustrative only, and the disclosed polypeptides, antibodies, and methods are not limited by the accuracy or inaccuracy of any such suggested mechanisms or modes of action or reasons for improvement.

[0022] Throughout this context, the description refers to polypeptides, antibodies, and methods for producing polypeptides and antibodies. Where this disclosure describes or claims features or embodiments relating to polypeptides or antibodies, such features or embodiments are equally applicable to methods for producing polypeptides or antibodies. Similarly, where this disclosure describes or claims features or embodiments relating to methods for producing polypeptides or antibodies, such features or embodiments are equally applicable to polypeptides or antibodies.

[0023] For clarity, it should be understood that certain features of the disclosed polypeptides, antibodies, and methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed polypeptides, antibodies, and methods described in relation to a single embodiment may also be provided separately or in any subcombination.

[0024] As used herein, the singular forms "a," "an," and "the" include the plural forms.

[0025] Where a list is presented, unless otherwise specified, each individual element of that list and every combination of that list should be understood to be a distinct embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0026] Various terms relating to the manner of description are used throughout this specification and the claims. Unless otherwise specified, such terms should be given their ordinary meanings in the art. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein.

[0027] The term “includes” is intended to include examples that are encompassed by the terms “essentially derived from” and “consisting of,” and similarly, the term “essentially derived from” is intended to include examples that are encompassed by the term “consisting of.”

[0028] Specific terms As used herein, “antibody” means an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment or derivative thereof, derived from any animal, including but not limited to sheep, rabbits, goats, mice, camels, and egg yolks.

[0029] 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 altered antibody-like structures. Engineered antibodies or antibody constructs are defined by the formula I:Z-(XY n -Z r ) p The antibody comprises an antibody-like structure having a polypeptide described herein, comprising the amino acid sequence of -X, wherein Z is a hinge comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102, X is a CH2 domain comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y is a linker, n is an integer of 1 or more, p is an integer of 2 or more, and r is an integer of 0 or 1. The antibody may be produced or modified by recombinant DNA or other synthetic techniques. The antibody may be an antibody isolated from a mammalian serum sample, an affinity-purified antibody, or any mixture thereof that exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. Also included are functional derivatives or "equivalents" of antibodies, such as single-chain antibodies and CDR graft antibodies. Also included are VHH antibodies, which may be monovalent or bivalent.

[0030] As used herein, “biotin” means biotin or any biotin derivative, and is not limited to substituted and unsubstituted biotins, as well as their analogs and derivatives, and similarly includes caproylamide biotin, biocitin, desthiobiotin, desthiobiocitin, iminobiotin, and substituted and unsubstituted derivatives of biotin sulfone.

[0031] As used herein, “coding sequence” or “coding polynucleotide” means an RNA or DNA molecule containing a nucleotide sequence that codes for a protein. The coding sequence may further include start and terminate signals operably linked to a regulatory element containing a promoter and a polyadenylation signal that can direct the expression of the nucleic acid molecule in cells of a target or mammal to which it is administered.

[0032] As used herein in the context of two or more nucleic acid molecules or polypeptide sequences, “identity” or “percent identity” means that the sequences have a specified percentage of residues that are the same across a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences across the specified region, determining the number of positions where identical residues exist in both sequences 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. If the two sequences are of different lengths, or if the alignment results in one or more attached ends and the specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequencing algorithm such as BLAST or BLAST 2.0.

[0033] As used herein, "fluorophore" means a compound that, when excited by exposure to light of a specific wavelength, emits light (fluoresces) at a wavelength different from the wavelength of exposure. Fluorophores may include substituents that modify the solubility, spectral properties, or physical properties of the fluorophore.

[0034] Examples of fluorophores include, but are not limited to, xanthenes, fluorescein, rhodamine, silicon rhodamine, rhodol, roseamine, carpopyranone, indole, indacene, borapoliazindacene, furan, benzofuran, cyanine, benzocyanine, benzopyrylene, pyrene, coumarin, styryl, squaline, resorphine, anthraquinone, acridine, benzophenoxazine, quinoline, quinazolinone, oligonucleotide-based fluorescent dyes, conjugated polymer dyes, and tandem dyes.

[0035] Examples of fluorophores suitable for use in the disclosed assays include 6-carboxyfluorescein (FAM), tetrachlorofluorescein (TET), tetramethylrhodamine (TMR), hexachlorofluorescein (HEX), JOE, 6-carboxy-X-rhodamine (ROX), CAL Fluor®, Pulsar®, Quasar®, Texas Red®, Texas Red-X, Cy®3, Cy®5, BODIPY® (boron-dipyromethene), Alexa Fluor® dyes (such as Alexa Fluor®488), and Alexa Fluor Plus dyes. DyLight® dyes (DyLight® 488, etc.), Oregon Green®, and NovaFluor® dyes (NovaFluor® Red 710, etc.), as well as Johnson, I. and Spence, MTZ (Eds.). (2010) The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11 thOther fluorophores listed in this edition include, but are not limited to, those mentioned.

[0036] As used herein, “immunospecifically binding” means the ability of a disclosed polypeptide or antibody to preferentially bind to its targets in a sample or environment containing a mixed population of molecules, without preferentially binding to other molecules.

[0037] As used herein, “label” means a drug that can be detected by, for example, ELISA, spectrophotometrics, flow cytometry, or microscopy. For example, a label can be bound (e.g., covalently or conjugated) to a nucleic acid molecule or protein (such as an antibody or its antigen-binding fragment), thereby enabling the detection of the 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 fluorescence and fluorescence-generating moieties (e.g., fluorophores).

[0038] Examples of labels include those that can be directly observed or measured, or those that can be observed or measured indirectly. Such labels include, but are not limited to, radioactive labels that can be measured with a radiation counter, pigments, dyes, or other colorants that can be visually observed or measured with a spectrophotometer, spin labels that can be measured with a spin labeling analyzer, and fluorescent moieties whose output signal is generated by the excitation of a suitable molecular adduct and can be visualized by excitation with light absorbed by the dye, or can be measured, for example, with a standard fluorophotometer or imaging system.

[0039] The label may be a luminescent substance such as a phosphorescent or fluorogen, a bioluminescent substance, a chemiluminescent substance whose output signal is generated by chemical modification of the signal compound, a metal-containing substance, or an enzyme that produces an enzyme-dependent secondary signal, such as the formation of a colored product from a colorless substrate. The label may also take the form of chemical, biochemical, or inert particles, including but not limited to colloidal gold, microspheres, quantum dots, or inorganic crystals such as nanocrystals or phosphorescent substances (see, for example, Beverloo, et al. Biochem. 203, 326-34 (1992)).

[0040] The term "labeling" can also refer to a "tag" or hapten that can selectively bind to the labeling molecule so that it can be used to generate a detectable signal when subsequently added. For example, biotin, iminobiotin, or desthiobiotin can be used as a tag, which can then be conjugated using avidin or streptavidin conjugate of horseradish peroxidase (HRP), and the presence of HRP can then be detected using a chromogenic substrate (e.g., tetramethylbenzidine) or a fluorescent substrate, such as Amplex® Red or Amplex® Gold (Thermo Fisher Scientific, Waltham, MA). In a similar manner, the tag can be a hapten or antigen (e.g., digoxigenin) which can be conjugated using an enzymatically, fluorescently, or radioactively labeled antibody.

[0041] Numerous labels are known to those skilled in the art, including particles, fluorescent dyes, haptens, enzymes, and their chromogenic substrates, fluorescence-generating substrates, and chemiluminescent substrates, as well as Johnson, I. and Johnson, I. and Spence, MTZ, (Eds.). (2010) The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Other markings listed in the edition include, but are not limited to, those listed in the edition.

[0042] As used herein, “nucleic acid molecule,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-strand description also defines the sequence of the complementary strand. Thus, 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 purposes as a given nucleic acid molecule. Thus, a nucleic acid molecule also encompasses substantially identical nucleic acid molecules and their complements.

[0043] 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 (both genomic and cDNA), RNA, or hybrids, and may 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 may have post-transcriptional chemical modifications. Nucleic acid molecules can be obtained by chemical synthesis or recombinant methods.

[0044] As used herein, “oligonucleotide-based fluorescent dyes” refers to nucleic acid nanostructures consisting of DNA-based scaffolds that precisely position fluorophores to manipulate their interactions and the overall fluorescent properties of their structure. Examples of oligonucleotide-based fluorescent dyes are described in International Patent Application Publication No. 2018 / 231805, including, but are not limited to, NovaFluor® dyes (Thermo Fisher Scientific, Waltham, MA).

[0045] As used herein, “peptide,” “protein,” or “polypeptide” may mean a linked sequence of amino acids, which may be natural, synthetic, or a combination of natural and synthetic modifications. Peptides, proteins, or polypeptides may have post-translational modifications.

[0046] As used herein, “vector” means a polynucleotide sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may contain or may contain one or more heterologous polynucleotide sequences.

[0047] Manipulated antibodies Recombinant antibodies offer the ability to manipulate benefits in a rational manner. One of the most important characteristics of an antibody is its sensitivity, i.e., the intensity of the signal it produces in a particular immunoassay. Highly sensitive antibodies have several advantages, such as the ability to detect low-abundance or rare targets and to detect proteins of interest in complex assays such as imaging. Antibody sensitivity is influenced by both increased signal and decreased background. Antibody sensitivity can be increased by affinity maturation of the antibody clone, which includes mutations in the antigen-binding site or paratope. However, affinity maturation must be adjusted for each antibody and is therefore not a scalable solution for dealing with a large number of antibodies. Furthermore, mutations in the paratope alter the antibody clone, essentially producing a new antibody.

[0048] This disclosure provides an approach to increase antibody sensitivity through rational modification of the antibody Fc region. Amplification or expansion of the entire Fc domain or a portion of the Fc domain provides additional binding sites for secondary antibodies, resulting in enhanced signaling. Previous studies have utilized Fc amplification for other purposes, such as increased antibody efficacy in therapeutic application through enhanced binding to Fc receptors, in the case of humanized antibodies. Antibody modification by adding one additional Fc domain (1TdFc) or two additional Fc domains (2TdFc) to the antibody has been found to result in insufficient expression with low yields and has aggregation and purity issues. To overcome the above limitations, this disclosure provides a rational design to avoid the shortcomings of previous studies by selectively adding additional CH2 domains (2TdCH2) instead of the entire Fc region. The 2TdCH2 design of the polypeptide and antibody provided herein overcomes the aforementioned challenges, and it was unexpectedly found that the 2TdCH2-modified antibodies provided herein possess yield, monomer purity, and stability comparable to wild-type antibodies. Two additional CH2 domains linked by a hinge and linker provide further epitopes for secondary antibody binding and additional lysine residues for conjugation. In addition, the 2TdCH2-modified antibodies of this disclosure provide a significant increase in sensitivity in multiple applications such as Western blotting and immunocytochemistry, enabling broader applicability of the antibodies. In addition to the advantages of non-conjugated antibodies, the 2TdCH2 modification also provides additional binding sites for different conjugation chemistry. Furthermore, it was unexpectedly found that the introduction of specific mutations, L234F, L235E, and P331S, referred to herein as “LFLEPS mutations,” into the modified scaffold reduced background binding of the antibody to Fc gamma receptors present on selected cells.Accordingly, the advantages of the manipulated antibodies of this disclosure include: i) at least 1.5-fold increased sensitivity; ii) at least 1.5-fold reduced background; ii) detection of low-abundance targets that are difficult to detect by other methods with standard antibodies; iii) increased coverage through the use of multiplexing; iv) increased conjugation sites for primary conjugates; and v) addressing the design challenges of expression, purity, and stability found in other recombinant antibodies.

[0049] In this specification, formula I:Z-(XY n -Z r ) p Disclosed is an isolated polypeptide comprising, essentially having, or consisting of the amino acid sequence -X, where Z is a hinge region, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1. In certain embodiments, Z-(XY n -Z r ) p The elements of the amino acid sequence of formula I, as defined herein as -X, are directly linked to one another without intervening amino acid sequences.

[0050] The hinge (Z) region may contain the amino acid sequence of SEQ ID NO: 18. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. Hinge (Z) may contain the amino acid sequence of SEQ ID NO: 48. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. Hinge (Z) may contain the amino acid sequence of SEQ ID NO: 102. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0051] 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.

[0052] The isolated polypeptide may contain one or more linkers (Y) containing the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. One or more linkers (Y) may contain GS. One or more linkers (Y) may contain GGS. One or more linkers (Y) may contain SEQ ID NO: 14. One or more linkers (Y) may contain SEQ ID NO: 38. One or more linkers may contain SEQ ID NO: 43. One or more linkers (Y) may contain sequences having at least 90% sequence identity to SEQ ID NO: 43. One or more linkers (Y) may contain sequences having at least 90% sequence identity to SEQ ID NO: 44. One or more linkers (Y) may contain sequences having at least 90% sequence identity to SEQ ID NO: 44.

[0053] An isolated polypeptide can contain one linker (Y) if n is 1. An isolated polypeptide can contain two linkers (Y) if n is 2. An isolated polypeptide can contain three linkers (Y) if n is 3. An isolated polypeptide can contain four linkers (Y) if n is 4. An isolated polypeptide can contain five linkers (Y) if n is 5.

[0054] The isolated polypeptide has a CH2-linker region ((XY n ) p The isolated polypeptide may contain two or more regions including ((XY) such that p is 2. n ) p ) may include three CH2-linker regions ((XY n ) p ) may include four CH2-linker regions ((XY n ) p ) may include five CH2-linker regions ((XY n ) p ) can include

[0055] The isolated polypeptide has a CH2-linker-hinge region ((XY n -Z r ) p The isolated polypeptide may contain two or more regions including ((XY) such that p is 2. n -Z r ) p ) may contain three CH2-linker-hinge regions ((XY n -Z r ) p) may contain four CH2-linker-hinge regions ((XY n -Z r ) p ) may contain five CH2-linker-hinge regions ((XY n -Z r ) p ) can include

[0056] In some embodiments, the isolated polypeptide is (XY n -Z r ) p It contains, essentially consists of, or is composed of, where 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, n is 1, r is 0 or 1, and p is 2.

[0057] In some embodiments, the isolated polypeptide is (XY n -Z r ) p It contains, essentially consists of, or is composed of, where 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 (linker) contains the amino acid sequence of SEQ ID NO: 44, n is 1, r is 0 or 1, and p is 2.

[0058] In some embodiments, the isolated polypeptide is (XY n -Z r ) p It contains, essentially consists of, or is composed of, where 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: 44, n is 1, r is 0 or 1, and p is 2.

[0059] In some embodiments, the isolated polypeptide is (XY n -Z r ) p It contains, essentially consists of, or is composed of, where 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, n is 1, r is 0 or 1, and p is 2.

[0060] In some embodiments, the isolated polypeptide contains 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 containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated polypeptide containing a CH3 domain contains SEQ ID NO: 52. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain an amino acid sequence having at least 90% identity to SEQ ID NO: 52. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain an amino acid sequence having at least 95% identity to SEQ ID NO: 52.

[0061] In some embodiments, the isolated polypeptide includes two additional CH2 domains at the N-terminus of the Fc domain, connected via a hinge and optionally one or more linkers, and contains the "LFLEPS" mutation in the lower hinge and CH2 domain. The "LFLEPS" mutation may include the following amino acid substitutions: L234F, L235E, and P331S. The isolated polypeptide includes two additional CH2 domains at the N-terminus of the Fc domain, connected via a hinge and optionally one or more linkers, and contains the "LFLEPS" mutation in the lower hinge and CH2 domain, and includes SEQ ID NO: 54. The isolated polypeptide includes two additional CH2 domains at the N-terminus of the Fc domain, connected via a hinge and optionally one or more linkers, and contains the "LFLEPS" mutation in the lower hinge and CH2 domain, and may contain an amino acid sequence having at least 90% identity to SEQ ID NO: 54. An isolated polypeptide containing two additional CH2 domains at the N-terminal end of an Fc domain connected via a hinge and optionally one or more linkers, and containing the "LFLEPS" mutation within the lower hinge and CH2 domains, may contain an amino acid sequence having at least 95% identity to SEQ ID NO: 54.

[0062] Any isolated polypeptide disclosed herein can be linked to a second polypeptide by one or more disulfide bonds, and the isolated polypeptide and the second polypeptide share the same amino acid sequence. One or more disulfide bonds can occur between the hinge of the isolated polypeptide and the hinge of the second polypeptide.

[0063] Any isolated polypeptide disclosed herein may further comprise a CH3 domain. In certain embodiments, the isolated polypeptide disclosed herein further comprises one or fewer CH3 domains. In some embodiments, the CH3 domain comprises the amino acid sequence of SEQ ID NO: 20. The CH3 domain may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. The CH3 domain may comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0064] In some embodiments, the isolated polypeptide containing the CH3 domain contains SEQ ID NO: 106. The isolated polypeptide containing the CH3 domain may contain an amino acid sequence having at least 90% identity with SEQ ID NO: 106. The isolated polypeptide containing the CH3 domain may contain an amino acid sequence having at least 95% identity with SEQ ID NO: 106.

[0065] In some embodiments, the isolated polypeptide containing the CH3 domain contains 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.

[0066] Furthermore, this specification provides antibodies comprising any isolated peptide disclosed herein ligated to a Fab domain. Fab may include a CH1 domain. The CH1 domain may include the amino acid sequence of SEQ ID NO: 17. The CH1 domain may include an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. The CH1 domain may include an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. Fab may include a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0067] In some embodiments, Fab may include a CH1 domain. The CH1 domain may include the amino acid sequence of SEQ ID NO: 100. The CH1 domain may include an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 100. The CH1 domain may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 100. Fab may include a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0068] In some embodiments, Fab may include a CH1 domain. The CH1 domain may include the amino acid sequence of SEQ ID NO: 160. The CH1 domain may include an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 160. The CH1 domain may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 160. Fab may include a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0069] In some embodiments, an antibody comprising any isolated peptide disclosed herein ligated to a Fab domain has at least a 1.5-fold reduction in background signal compared to the corresponding wild-type antibody. In some embodiments, the reduction in background signal is 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, or at least 5-fold compared to the corresponding wild-type antibody.

[0070] In some embodiments, an antibody comprising any isolated peptide disclosed herein ligated to a Fab domain has at least a 1.5-fold increase in sensitivity compared to the corresponding wild-type antibody. In some embodiments, the increase in sensitivity is 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, at least 9-fold, at least 9.5-fold, or at least 10-fold compared to the corresponding wild-type antibody.

[0071] In some embodiments, an antibody comprising any isolated peptide disclosed herein ligated to a Fab domain has at least a 1.5-fold increase in signal enhancement compared to the corresponding wild-type antibody. In some embodiments, the increase in signal enhancement is 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, at least 9-fold, at least 9.5-fold, or at least 10-fold compared to the corresponding wild-type antibody.

[0072] The antibody Fab can immunospecifically bind to one or more target molecules. The Fab can immunospecifically bind to Parkin, OCT4, IDO1, FLAG-Tag, pERK1 / 2, PSD-95, or TIGIT, or the Fab can be a rabbit anti-mouse (RAM) IgG secondary antibody.

[0073] In some embodiments, Fab binds immunospecifically to parkin. Fab that binds immunospecifically to parkin may include a Fab heavy chain comprising a heavy chain complementarity determination 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. In certain embodiments throughout this disclosure, CDRs are defined according to the IMGT system.

[0074] A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 7. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 7. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 7.

[0075] Fab that binds immunospecifically to parkin may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 9. Fab that binds immunospecifically to parkin may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9. Fab that binds immunospecifically to parkin may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9.

[0076] Fab, which binds immunospecifically to parkin, may include a Fab light chain comprising 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.

[0077] A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 16. A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0078] A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 12. A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12. A Fab that immune-specifically binds to parkin may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 12.

[0079] Fab, which binds immunospecifically to parkin, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a Fab light chain comprising 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.

[0080] A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 7, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 16. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0081] A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 12. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 12. A Fab that immune-specifically binds to parkin may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 12.

[0082] In some embodiments, an antibody containing Fab that immunospecifically binds to parkin contains the amino acid sequence of SEQ ID NO: 3. An antibody containing Fab that immunospecifically binds to parkin may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3. An antibody containing Fab that immunospecifically binds to parkin may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3.

[0083] In some embodiments, the Fab binds immunospecifically to OCT4. The Fab that binds immunospecifically to OCT4 may include 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.

[0084] A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 32. A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32. A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32.

[0085] A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 34. A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34. A Fab that binds immunospecifically to OCT4 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34.

[0086] Fab, which binds immunospecifically to OCT4, may include a Fab light chain comprising a light chain complementarity determination 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.

[0087] A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 40. A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40. A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40.

[0088] A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 36. A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36. A Fab that binds immunospecifically to OCT4 may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 36.

[0089] Fab, which binds immunospecifically to OCT4, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a Fab light chain comprising a light chain complementarity determination 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.

[0090] A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 32, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 40. A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 40. A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0091] A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 36. A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 36. A Fab that immune-specifically binds to OCT4 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 36.

[0092] In some embodiments, an antibody containing Fab that immunospecifically binds to OCT4 contains the amino acid sequence of SEQ ID NO: 28. An isolated polypeptide containing Fab that immunospecifically binds to OCT4 may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28. An isolated polypeptide containing Fab that immunospecifically binds to OCT4 may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28.

[0093] In some embodiments, the Fab binds immunospecifically to IDO1. The Fab that binds immunospecifically to IDO1 may include 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.

[0094] A Fab that binds immunospecifically to IDO1 may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 65. A Fab that binds immunospecifically to IDO1 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65. A Fab that binds immunospecifically to IDO1 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65.

[0095] Fab, which binds immunospecifically to IDO1, may include a 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.

[0096] A Fab that binds immunospecifically to IDO1 may include a Fab light chain containing a variable region with an amino acid sequence of SEQ ID NO: 71. A Fab that binds immunospecifically to IDO1 may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. A Fab that binds immunospecifically to IDO1 may include a Fab light chain containing a variable domain with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0097] Fab, which binds immunospecifically to IDO1, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a 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.

[0098] A Fab that immune-specifically binds to IDO1 may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 65, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 71. A Fab that immune-specifically binds to IDO1 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71. A Fab that immune-specifically binds to IDO1 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0099] In some embodiments, an antibody containing Fab that immunospecifically binds to IDO1 contains the amino acid sequence of SEQ ID NO: 79. An isolated polypeptide containing Fab that immunospecifically binds to IDO1 may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79. An isolated polypeptide containing Fab that immunospecifically binds to IDO1 may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 79.

[0100] In some embodiments, the Fab immunospecifically binds to the FLAG-tag. The Fab that immunospecifically binds to the FLAG-tag may include a heavy chain complementarity determination 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.

[0101] A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 162. A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 162. A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162.

[0102] Fab that binds immunospecifically to the FLAG-tag may include a Fab light chain comprising 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.

[0103] A Fab that binds immunospecifically to a FLAG-tag may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 167. A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 167. A Fab that binds immunospecifically to a FLAG-tag may include a Fab light chain containing a variable domain with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 167.

[0104] A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain comprising a heavy chain complementarity determination 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; and a Fab light chain comprising 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.

[0105] A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 162, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 167. A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 167. A Fab that binds immunospecifically to a FLAG-tag may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 167.

[0106] In some embodiments, an antibody containing Fab that immunospecifically binds to the FLAG-tag contains the amino acid sequence of SEQ ID NO: 175. An isolated polypeptide containing Fab that immunospecifically binds to the FLAG-tag may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 175. An isolated polypeptide containing Fab that immunospecifically binds to the FLAG-tag may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 175.

[0107] 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, which is RAM, may include 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.

[0108] A RAM-based Fab may contain a Fab light chain that includes a variable region containing the amino acid sequence of SEQ ID NO: 126. A RAM-based Fab may contain a Fab heavy chain that includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126. A RAM-based Fab may contain a Fab heavy chain that includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126.

[0109] The RAM, Fab, may include 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.

[0110] A RAM-based Fab may contain a Fab light chain that includes a variable region containing the amino acid sequence of SEQ ID NO: 131. A RAM-based Fab may contain a Fab light chain that includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 131. A RAM-based Fab may contain a Fab light chain that includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 131.

[0111] The RAM, Fab, may include a Fab heavy chain comprising a heavy chain complementarity determination 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; and 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.

[0112] A RAM-based Fab may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 126, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 131. A RAM-based Fab may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 131. A RAM-based Fab may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 131.

[0113] In some embodiments, the antibody containing Fab, which is RAM, contains the amino acid sequence of SEQ ID NO: 139. The isolated polypeptide containing RAM may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 139. The isolated polypeptide containing RAM may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 139.

[0114] In some embodiments, Fab immunospecifically binds to pERK1 / 2. Fab immunospecifically binding to pERK1 / 2 may include 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.

[0115] Fab that binds immunospecifically to pERK1 / 2 may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 84. Fab that binds immunospecifically to pERK1 / 2 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84. Fab that binds immunospecifically to pERK1 / 2 may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84.

[0116] Fab, which binds immunospecifically to pERK1 / 2, may include a Fab light chain comprising a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, LCDR2 containing the amino acid sequence of KVS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0117] Fab that binds immunospecifically to pERK1 / 2 may include a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 90. Fab that binds immunospecifically to pERK1 / 2 may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 90. Fab that binds immunospecifically to pERK1 / 2 may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90.

[0118] Fab, which binds immunospecifically to pERK1 / 2, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a Fab light chain comprising a light chain complementarity determination 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.

[0119] A Fab that immune-specifically binds to pERK1 / 2 may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 84, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 90. A Fab that immune-specifically binds to pERK1 / 2 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 90. A Fab that immune-specifically binds to pERK1 / 2 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 90.

[0120] In some embodiments, an antibody containing Fab that immunospecifically binds to pERK1 / 2 contains the amino acid sequence of SEQ ID NO: 98. An isolated polypeptide containing Fab that immunospecifically binds to pERK1 / 2 may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 98. An isolated polypeptide containing Fab that immunospecifically binds to pERK1 / 2 may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 98.

[0121] In some embodiments, the Fab binds immunospecifically to PSD-95. The Fab that binds immunospecifically to PSD-95 may include 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.

[0122] A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 110. A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region having at least 90% sequence identity with SEQ ID NO: 110. A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region having at least 95% sequence identity with SEQ ID NO: 110.

[0123] Fab, which binds immunospecifically to PSD-95, may include a Fab light chain comprising 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.

[0124] A Fab that binds immunospecifically to PSD-95 may include a Fab light chain containing a variable region with an amino acid sequence of SEQ ID NO: 115. A Fab that binds immunospecifically to PSD-95 may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. A Fab that binds immunospecifically to PSD-95 may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 115.

[0125] Fab, which binds immunospecifically to PSD-95, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a Fab light chain comprising a light chain complementarity determination 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.

[0126] A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 110, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 115. A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 115. A Fab that binds immunospecifically to PSD-95 may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 115.

[0127] In some embodiments, an antibody containing Fab that immunospecifically binds to PSD-95 contains the amino acid sequence of SEQ ID NO: 123. An isolated polypeptide containing Fab that immunospecifically binds to PSD-95 may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123. An isolated polypeptide containing Fab that immunospecifically binds to PSD-95 may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 123.

[0128] In some embodiments, the Fab binds immunospecifically to TIGIT. The Fab that binds immunospecifically to TIGIT may include a heavy chain complementarity determination 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.

[0129] A Fab that binds immunospecifically to TIGIT may include a Fab heavy chain containing a variable region with the amino acid sequence of SEQ ID NO: 145. A Fab that binds immunospecifically to TIGIT may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145. A Fab that binds immunospecifically to TIGIT may include a Fab heavy chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145.

[0130] Fab, which binds immunospecifically to TIGIT, may include a Fab light chain comprising 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.

[0131] A Fab that binds immunospecifically to TIGIT may include a Fab light chain containing a variable region with an amino acid sequence of SEQ ID NO: 150. A Fab that binds immunospecifically to TIGIT may include a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 150. A Fab that binds immunospecifically to TIGIT may include a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 150.

[0132] Fab, which binds immunospecifically to TIGIT, may include a Fab heavy chain comprising a heavy chain complementarity determination 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, and a Fab light chain comprising 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.

[0133] A Fab that immune-specifically binds to TIGIT may include a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 145, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 150. A Fab that immune-specifically binds to TIGIT may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 150. A Fab that immune-specifically binds to TIGIT may include a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 150.

[0134] In some embodiments, the Fab that immunospecifically binds to TIGIT contains the amino acid sequence of SEQ ID NO: 157. The isolated polypeptide containing the Fab that immunospecifically binds to TIGIT may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 157. The isolated polypeptide containing the Fab that immunospecifically binds to TIGIT may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 157.

[0135] Any antibody disclosed herein may be an antibody having isotype IgG.

[0136] In some embodiments, the isolated polypeptide or antibody further comprises a label.

[0137] In this specification, formula I:Z-(XY n -Z r ) p A nucleic acid molecule is disclosed which comprises, essentially comprises, or comprises a polynucleotide encoding an isolated polypeptide comprising, essentially comprising, or comprising the amino acid sequence -X, wherein Z is a hinge, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1.

[0138] A nucleic acid molecule may contain a polynucleotide encoding a hinge (Z) that includes the amino acid sequence of SEQ ID NO: 18. The polynucleotide may encode a hinge (Z) that includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The polynucleotide may encode a hinge (Z) that includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the hinge (Z) includes SEQ ID NO: 22.

[0139] Nucleic acid molecules are (XY n -Z r ) pThe polynucleotide may include a polynucleotide encoding a polypeptide that contains, is essentially derived from, or consists of, the hinge(Z) comprising 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 to SEQ ID NO: 48. The polynucleotide may encode a hinge(Z) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 48. In some embodiments, the polynucleotide encoding hinge(Z) comprises SEQ ID NO: 51.

[0140] Nucleic acid molecules are (XY n -Z r The polynucleotide may include a polynucleotide encoding a polypeptide containing, essentially derived from, or consisting of ) where hinge(Z) contains the amino acid sequence of SEQ ID NO: 102. The polynucleotide may encode a hinge(Z) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 102. The polynucleotide may encode a hinge(Z) containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, the polynucleotide encoding hinge(Z) contains SEQ ID NO: 101.

[0141] (XY n -Z r ) p A polynucleotide encoding a polypeptide containing, essentially derived from, or consisting of can encode a CH2 domain (X) containing the amino acid sequence of SEQ ID NO: 19. The polynucleotide can encode a CH2 domain (X) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. The polynucleotide can encode a CH2 domain (X) containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the CH2 domain (X) contains SEQ ID NO: 23 or SEQ ID NO: 178.

[0142] (XY n -Z r ) p A nucleic acid molecule encoding a polypeptide containing, essentially derived from, or consisting of may include a polynucleotide encoding a CH2 domain (X) containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide may encode a CH2 domain (X) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 49. The polynucleotide may encode a CH2 domain (X) containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 49. In some embodiments, the polynucleotide encoding the CH2 domain (X) includes SEQ ID NO: 62.

[0143] (XY n -Z r ) p A polynucleotide encoding a polypeptide containing, essentially derived from, or consisting of ista can encode a CH2 domain (X) containing the amino acid sequence of SEQ ID NO: 102. The polynucleotide can encode a CH2 domain (X) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 102. The polynucleotide can encode a CH2 domain (X) containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, the polynucleotide encoding the CH2 domain (X) contains SEQ ID NO: 101.

[0144] (XY n -Z r ) pA nucleic acid molecule encoding a polypeptide containing, essentially derived from, or composed of a polypeptide may include a polynucleotide encoding an isolated polypeptide containing one or more linkers (Y) containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide may encode one or more linkers (Y) containing GS. A polynucleotide may encode one or more linkers (Y) containing GGS. A polynucleotide may encode one or more linkers (Y) containing SEQ ID NO: 14. A polynucleotide may encode one or more linkers (Y) containing SEQ ID NO: 38. A polynucleotide may encode one or more linkers (Y) containing SEQ ID NO: 43. A polynucleotide may encode one or more linkers (Y) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43. A polynucleotide may encode one or more linkers (Y) containing SEQ ID NO: 44. A polynucleotide may encode one or more linkers (Y) containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, the polynucleotide encoding one or more linkers (Y) includes SEQ ID NO: 46.

[0145] A polynucleotide can encode an isolated polypeptide containing one linker (Y), such that n is 1. A polynucleotide can encode an isolated polypeptide containing two linkers (Y), such that n is 2. A polynucleotide can encode an isolated polypeptide containing three linkers (Y), such that n is 3. A polynucleotide can encode an isolated polypeptide containing four linkers (Y), such that n is 4. A polynucleotide can encode an isolated polypeptide containing five linkers (Y), such that n is 5.

[0146] Polynucleotides have a CH2-linker region ((XY n) p ) can encode an isolated polypeptide comprising two or more regions. The polynucleotide can comprise two CH2-linker regions ((X-Y such that p is 2 n ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise three CH2-linker regions ((X-Y such that p is 3 n ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise four CH2-linker regions ((X-Y such that p is 4 n ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise five CH2-linker regions ((X-Y such that p is 5 n ) p ) can encode an isolated polypeptide comprising.

[0147] The polynucleotide can comprise a CH2-linker-hinge region ((X-Y such that p is 2 or more n -Z r ) p ) can encode an isolated polypeptide comprising two or more regions. The polynucleotide can comprise two CH2-linker-hinge regions ((X-Y such that p is 2 n -Z r ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise three CH2-linker-hinge regions ((X-Y such that p is 3 n -Z r ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise four CH2-linker-hinge regions ((X-Y such that p is 4 n -Z r ) p ) can encode an isolated polypeptide comprising. The polynucleotide can comprise five CH2-linker-hinge regions ((X-Y such that p is 5 n -Z r )p It can encode isolated polypeptides containing ).

[0148] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide which comprises, essentially, or consists of, an isolated polypeptide comprising 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, where 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.

[0149] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide which comprises, essentially, or consists of, an isolated polypeptide comprising 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, where 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.

[0150] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an isolated polypeptide which comprises, or is essentially composed of, or is composed of, an isolated polypeptide comprising Z (hinge) containing the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) containing the amino acid sequence of SEQ ID NO: 104, and Y (linker) containing the amino acid sequence of SEQ ID NO: 44, where 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.

[0151] A polynucleotide can encode an isolated polypeptide containing two additional CH2 domains at the N-terminus of an Fc domain, linked via a hinge and optionally one or more linkers, and the isolated polypeptide contains the amino acid sequence of SEQ ID NO: 52. In some embodiments, a polynucleotide encodes an isolated polypeptide containing two additional CH2 domains at the N-terminus of an Fc domain, linked via a hinge and optionally one or more linkers, and the isolated polypeptide contains SEQ ID NO: 52. A polynucleotide can encode an isolated polypeptide containing two additional CH2 domains at the N-terminus of an Fc domain, linked via a hinge and optionally one or more linkers, and the isolated polypeptide contains an amino acid sequence having at least 90% identity to SEQ ID NO: 52. A polynucleotide can encode an isolated polypeptide containing two additional CH2 domains at the N-terminus of an Fc domain, linked via a hinge and optionally one or more linkers, and the isolated polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 52. In some embodiments, a polynucleotide encoding an isolated polypeptide containing a hinge and two additional CH2 domains attached to the N-terminus of the Fc domain via one or more linkers, includes SEQ ID NO: 53.

[0152] In some embodiments, the polynucleotide encodes an isolated polypeptide comprising a hinge and two additional CH2 domains at the N-terminus of an Fc domain linked via one or more linkers of choice, and containing the “LFLEPS” mutation in the lower hinge and CH2 domain. In some embodiments, the “LFLEPS” mutation may include the following amino acid substitutions: L234F, L235E, and P331S. In some embodiments, the polynucleotide encodes an isolated polypeptide comprising a hinge and two additional CH2 domains at the N-terminus of an Fc domain linked via one or more linkers of choice, and containing the “LFLEPS” mutation in the lower hinge and CH2 domain, and the isolated polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 54. The polynucleotide can encode an isolated polypeptide comprising a hinge and two additional CH2 domains at the N-terminus of an Fc domain linked via one or more linkers of choice, and containing the “LFLEPS” mutation in the lower hinge and CH2 domain, and the isolated polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 54. A polynucleotide can encode an isolated polypeptide comprising a hinge and two additional CH2 domains at the N-terminus of an Fc domain linked via one or more linkers of choice, and containing the "LFLEPS" mutation within the lower hinge and CH2 domain, the isolated polypeptide having an amino acid sequence having at least 95% identity to SEQ ID NO: 54. In some embodiments, a polynucleotide encoding an isolated polypeptide comprising a hinge and two additional CH2 domains at the N-terminus of an Fc domain linked via one or more linkers of choice, and containing the "LFLEPS" mutation within the lower hinge and CH2 domain, comprises SEQ ID NO: 55.

[0153] A polynucleotide can encode any isolated polypeptide disclosed herein, and can further encode a second polypeptide, the isolated polypeptide and the second polypeptide sharing the same amino acid sequence.

[0154] A polynucleotide can encode any isolated polypeptide disclosed herein, further comprising 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 to SEQ ID NO: 20. The polynucleotide can encode a CH3 domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the CH3 domain comprises SEQ ID NO: 24 or SEQ ID NO: 179.

[0155] In some embodiments, the polynucleotide encodes an isolated polypeptide containing a CH3 domain, and the isolated polypeptide comprises SEQ ID NO: 106. The polynucleotide can encode an isolated polypeptide containing a CH3 domain, and the isolated polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 106. The polynucleotide can encode an isolated polypeptide containing a CH3 domain, and the isolated polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 106. In some embodiments, the polynucleotide encoding an isolated polypeptide containing a CH3 domain comprises SEQ ID NO: 105.

[0156] In some embodiments, the polynucleotide encodes an isolated polypeptide containing a CH3 domain, and the isolated polypeptide includes SEQ ID NO: 141. The polynucleotide can encode an isolated polypeptide containing a CH3 domain, and the isolated polypeptide contains an amino acid sequence having at least 90% identity to SEQ ID NO: 141. The polynucleotide can encode an isolated polypeptide containing a CH3 domain, and the isolated polypeptide contains an amino acid sequence having at least 95% identity to SEQ ID NO: 141. In some embodiments, the polynucleotide encoding an isolated polypeptide containing a CH3 domain includes SEQ ID NO: 140.

[0157] A polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, further comprising Fab. A polynucleotide can encode a Fab comprising a CH1 domain. A polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 17. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. A polynucleotide encoding a CH1 domain may include SEQ ID NO: 21, SEQ ID NO: 176, or SEQ ID NO: 177. A polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0158] A polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, further comprising Fab. A polynucleotide can encode a Fab comprising a CH1 domain. A polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 100. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 100. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 100. A polynucleotide encoding a CH1 domain may include SEQ ID NO: 99. A polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0159] A polynucleotide can encode any antibody comprising any isolated polypeptide disclosed herein, further comprising Fab. A polynucleotide can encode a Fab comprising a CH1 domain. A polynucleotide can encode a CH1 domain comprising the amino acid sequence of SEQ ID NO: 160. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 160. A polynucleotide can encode a CH1 domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 160. A polynucleotide encoding a CH1 domain may include SEQ ID NO: 159. A polynucleotide can encode a Fab comprising a Fab heavy chain and a Fab light chain that pair to form a binding domain.

[0160] A polynucleotide can encode a Fab that immunospecifically binds to one or more target molecules. The polynucleotide can encode a Fab that immunospecifically binds to Parkin, OCT4, IDO1, FLAG-Tag, pERK1 / 2, PSD-95, or TIGIT, or the Fab can be a rabbit anti-mouse (RAM) IgG secondary antibody.

[0161] In some embodiments, the polynucleotide can encode a Fab that binds immunospecifically to parkin. The polynucleotide can encode a Fab that binds immunospecifically to parkin, and the Fab comprises 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.

[0162] A polynucleotide can encode Fab, which binds immunospecifically to parkin, and Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 7. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 7. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 7.

[0163] A polynucleotide can encode a Fab that binds immunospecifically to parkin, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 9. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 9. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 9. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to parkin, the polynucleotide comprises SEQ ID NO: 8.

[0164] The polynucleotide can encode Fab, which immunospecifically binds to parkin, and Fab comprises a Fab light chain containing a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 13, LCDR2 containing the amino acid sequence RAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0165] A polynucleotide can encode Fab, which immunospecifically binds to parkin, and Fab comprises a Fab light chain containing a variable region having the amino acid sequence of SEQ ID NO: 16. A polynucleotide can encode a Fab light chain containing a variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16. A polynucleotide can encode a Fab light chain containing a variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 16.

[0166] A polynucleotide can encode a Fab that binds immunospecifically to parkin, and the Fab comprises a Fab light chain containing a variable region having the amino acid sequence of SEQ ID NO: 12. A polynucleotide can encode a Fab light chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 12. A polynucleotide can encode a Fab light chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 12. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to parkin, the polynucleotide comprises SEQ ID NO: 11.

[0167] A polynucleotide can encode Fab, which binds immunospecifically to parkin. Fab comprises a Fab heavy chain, which includes a heavy chain complementarity determination 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; and 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.

[0168] A polynucleotide can encode Fab, which binds immunospecifically to parkin, and Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 7, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 16. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0169] A polynucleotide can encode a Fab that immunospecifically binds to parkin, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 12. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to parkin, the polynucleotide comprises SEQ ID NO: 8 and SEQ ID NO: 11.

[0170] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to parkin, wherein the Fab contains the amino acid sequence of SEQ ID NO: 3. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to parkin, the polynucleotide contains SEQ ID NO: 2.

[0171] In some embodiments, the polynucleotide can encode a Fab that immunospecifically binds to OCT4. The polynucleotide can encode a Fab that immunospecifically binds to OCT4, and the Fab includes 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.

[0172] A polynucleotide can encode a Fab that binds immunospecifically to OCT4, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 32. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 32. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 32.

[0173] A polynucleotide can encode a Fab that binds immunospecifically to OCT4, and the Fab comprises a Fab heavy chain that includes a variable region containing the amino acid sequence of SEQ ID NO: 34. A polynucleotide can encode a Fab heavy chain that includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34. A polynucleotide can encode a Fab heavy chain that includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to OCT4, the polynucleotide comprises SEQ ID NO: 33.

[0174] The polynucleotide can encode Fab, which immunospecifically binds to OCT4, and Fab comprises a Fab light chain containing a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 37, LCDR2 containing the amino acid sequence GAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0175] A polynucleotide can encode a Fab that binds immunospecifically to OCT4, and the Fab comprises a Fab light chain containing a variable region having the amino acid sequence of SEQ ID NO: 40. A polynucleotide can encode a Fab light chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 40. A polynucleotide can encode a Fab light chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 40.

[0176] A polynucleotide can encode a Fab that immunospecifically binds to OCT4, and the Fab includes a Fab light chain containing the amino acid sequence of SEQ ID NO: 36. A polynucleotide can encode a Fab light chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 36. A polynucleotide can encode a Fab light chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 36. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to OCT4, the polynucleotide includes SEQ ID NO: 35.

[0177] A polynucleotide can encode Fab, which binds immunospecifically to OCT4. Fab comprises a Fab heavy chain, which includes a heavy chain complementarity determination 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; and a Fab light chain, which includes a light chain complementarity determination 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.

[0178] A polynucleotide can encode Fab, which binds immunospecifically to OCT4, and Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 32, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 40. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 40. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0179] A polynucleotide can encode a Fab that immunospecifically binds to OCT4, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 36. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 36. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 34, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to OCT4, the polynucleotide includes SEQ ID NO: 33 and SEQ ID NO: 35.

[0180] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to OCT4, wherein the Fab contains the amino acid sequence of SEQ ID NO: 28. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to OCT4, the polynucleotide contains SEQ ID NO: 27.

[0181] In some embodiments, the polynucleotide can encode a Fab that binds immunospecifically to IDO1. The polynucleotide can encode a Fab that binds immunospecifically to IDO1, and the Fab comprises 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.

[0182] A polynucleotide can encode a Fab that binds immunospecifically to IDO1, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 64. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 64. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 64.

[0183] The polynucleotide can encode Fab, which immunospecifically binds to IDO1, and Fab comprises a Fab light chain containing a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 72, LCDR2 containing the amino acid sequence of YAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 73.

[0184] A polynucleotide can encode a Fab that binds immunospecifically to IDO1, and the Fab comprises a Fab light chain containing a variable region having the amino acid sequence of SEQ ID NO: 71. A polynucleotide can encode a Fab light chain containing a variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 71. A polynucleotide can encode a Fab light chain containing a variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 71.

[0185] A polynucleotide can encode Fab, which immunospecifically binds to IDO1. Fab comprises a Fab heavy chain, which includes a heavy chain complementarity determination 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; and a Fab light chain, which includes 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.

[0186] A polynucleotide can encode a Fab that immunospecifically binds to IDO1, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 65, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 71. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 65, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 65, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, a polynucleotide encoding a Fab that immunospecifically binds to IDO1 includes SEQ ID NO: 64 and SEQ ID NO: 70.

[0187] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to IDO1, wherein the Fab contains the amino acid sequence of SEQ ID NO: 79. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 79. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to IDO1, the polynucleotide contains SEQ ID NO: 78.

[0188] A polynucleotide can encode a Fab that binds immunospecifically to a FLAG-tag, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 162. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 162. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 162.

[0189] The polynucleotide can encode Fab, which immunospecifically binds to the FLAG-tag, and Fab comprises a Fab light chain containing a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 168, LCDR2 containing the amino acid sequence of YAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 169.

[0190] A polynucleotide can encode a Fab that binds immunospecifically to a FLAG-tag, and the Fab includes a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 167. A polynucleotide can encode a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 167. A polynucleotide can encode a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 167.

[0191] The polynucleotide can encode Fab, which binds immunospecifically to the FLAG-tag. Fab comprises a Fab heavy chain, which includes 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; and a Fab light chain, which includes 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.

[0192] A polynucleotide can encode a Fab that binds immunospecifically to a FLAG-tag, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 162, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 167. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 162, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 167. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 167. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to a FLAG-tag, the polynucleotide includes SEQ ID NO: 181 and SEQ ID NO: 166.

[0193] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to a FLAG-tag, wherein the Fab contains the amino acid sequence of SEQ ID NO: 175. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 175. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 175. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to a FLAG-tag, the polynucleotide contains SEQ ID NO: 174.

[0194] In some embodiments, the polynucleotide can encode Fab, which is a rabbit anti-mouse IgG secondary antibody (RAM). The polynucleotide can encode Fab, which is RAM, and Fab comprises a 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.

[0195] A polynucleotide can encode Fab, which is a RAM, and Fab comprises a Fab heavy chain, which includes a variable region containing the amino acid sequence of SEQ ID NO: 126. A polynucleotide can encode a Fab heavy chain, which includes a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 126. A polynucleotide can encode a Fab heavy chain, which includes a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126.

[0196] A polynucleotide can encode Fab, which is a RAM, and Fab comprises a Fab light chain comprising a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 132, LCDR2 containing the amino acid sequence of GAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 133.

[0197] A polynucleotide can encode Fab, which is a RAM, and Fab includes a Fab light chain that includes a variable region containing the amino acid sequence of SEQ ID NO: 131. A polynucleotide can encode a Fab light chain that includes a variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 131. A polynucleotide can encode a Fab light chain that includes a variable region containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 131.

[0198] A polynucleotide can encode Fab, which is a RAM, and Fab comprises a Fab heavy chain comprising a heavy chain complementarity determination 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, and 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.

[0199] A polynucleotide can encode a RAM called Fab, where Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 126, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 131. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 126, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 131. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 131. In some embodiments, a polynucleotide encoding an immunospecifically binding RAM called Fab comprises SEQ ID NO: 125 and SEQ ID NO: 130.

[0200] In some embodiments, the polynucleotide can encode an antibody containing Fab, which is RAM, and Fab contains the amino acid sequence of SEQ ID NO: 139. The polynucleotide can encode Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 139. The polynucleotide can encode Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 139. In some embodiments, in a polynucleotide encoding Fab, which is RAM, the polynucleotide contains SEQ ID NO: 138.

[0201] In some embodiments, the polynucleotide can encode a Fab that immunospecifically binds to pERK1 / 2. The polynucleotide can encode a Fab that immunospecifically binds to pERK1 / 2, and the Fab comprises a Fab heavy chain comprising 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.

[0202] A polynucleotide can encode a Fab that binds immunospecifically to pERK1 / 2, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 84. A polynucleotide can encode a Fab heavy chain containing a variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84. A polynucleotide can encode a Fab heavy chain containing a variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84.

[0203] The polynucleotide can encode Fab, which immunospecifically binds to pERK1 / 2, and Fab comprises a Fab light chain containing a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, LCDR2 containing the amino acid sequence of KVS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0204] A polynucleotide can encode a Fab that binds immunospecifically to pERK1 / 2, and the Fab comprises a Fab light chain containing a variable region with the amino acid sequence of SEQ ID NO: 90. A polynucleotide can encode a Fab light chain containing a variable region with an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 90. A polynucleotide can encode a Fab light chain containing a variable region with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 90.

[0205] A polynucleotide can encode Fab, which immunospecifically binds to pERK1 / 2. Fab comprises a Fab heavy chain containing 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; and 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 KVS, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 92.

[0206] A polynucleotide can encode a Fab that immunospecifically binds to pERK1 / 2, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 84, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 90. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 90. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 84, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 90. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to pERK1 / 2, the polynucleotide includes SEQ ID NO: 83 and SEQ ID NO: 89.

[0207] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to pERK1 / 2, wherein the Fab contains the amino acid sequence of SEQ ID NO: 98. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 98. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to pERK1 / 2, the polynucleotide contains SEQ ID NO: 97.

[0208] In some embodiments, the polynucleotide can encode a Fab that binds immunospecifically to PSD-95. The polynucleotide can encode a Fab that binds immunospecifically to PSD-95, and the Fab comprises 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.

[0209] A polynucleotide can encode a Fab that binds immunospecifically to PSD-95, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 110. A polynucleotide can encode a Fab heavy chain containing a variable region having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110. A polynucleotide can encode a Fab heavy chain containing a variable region having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110.

[0210] The polynucleotide can encode a Fab that immunospecifically binds to PSD-95, and the Fab comprises a Fab light chain, which comprises 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.

[0211] The polynucleotide can encode a Fab that immunospecifically binds to PSD-95, and the Fab comprises a Fab light chain which comprises a variable region comprising the amino acid sequence of SEQ ID NO: 115. The polynucleotide can encode a Fab light chain which comprises a variable region comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 115. The polynucleotide can encode a Fab light chain which comprises a variable region comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 115.

[0212] The polynucleotide can encode a Fab that immunospecifically binds to PSD-95, and the Fab comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain 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, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 113, and the Fab light chain comprises 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.

[0213] A polynucleotide can encode a Fab that immunospecifically binds to PSD-95, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 110, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 115. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 110, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 115. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 110, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 115. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to PSD-95, the polynucleotide comprises SEQ ID NO: 125 and SEQ ID NO: 130.

[0214] In some embodiments, the polynucleotide can encode an antibody containing a Fab that immunospecifically binds to PSD-95, wherein the Fab contains the amino acid sequence of SEQ ID NO: 123. The polynucleotide can encode a Fab containing an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123. The polynucleotide can encode a Fab containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 123. In some embodiments, in a polynucleotide encoding a Fab that immunospecifically binds to PSD-95, the polynucleotide contains SEQ ID NO: 122.

[0215] In some embodiments, the polynucleotide can encode a Fab that binds immunospecifically to TIGIT. The polynucleotide can encode a Fab that binds immunospecifically to TIGIT, and the Fab 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.

[0216] A polynucleotide can encode a Fab that binds immunospecifically to TIGIT, and the Fab comprises a Fab heavy chain containing a variable region having the amino acid sequence of SEQ ID NO: 145. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 145. A polynucleotide can encode a Fab heavy chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 145.

[0217] The polynucleotide can encode Fab, which binds immunospecifically to TIGIT, and Fab comprises a Fab light chain containing a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 151, LCDR2 containing the amino acid sequence of SAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 152.

[0218] A polynucleotide can encode a Fab that binds immunospecifically to TIGIT, and the Fab comprises a Fab light chain containing a variable region having the amino acid sequence of SEQ ID NO: 150. A polynucleotide can encode a Fab light chain containing a variable region having at least 90% sequence identity with respect to SEQ ID NO: 150. A polynucleotide can encode a Fab light chain containing a variable region having at least 95% sequence identity with respect to SEQ ID NO: 150.

[0219] A polynucleotide can encode Fab, which binds immunospecifically to TIGIT. Fab comprises a Fab heavy chain, which includes a heavy chain complementarity determination 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; and a Fab light chain, which includes 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.

[0220] A polynucleotide can encode a Fab that binds immunospecifically to TIGIT, and the Fab comprises a Fab heavy chain containing a variable region containing the amino acid sequence of SEQ ID NO: 145, and a Fab light chain containing a variable region containing the amino acid sequence of SEQ ID NO: 150. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145, and a Fab light chain containing a variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 150. A polynucleotide can encode a Fab heavy chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145, and a Fab light chain containing a variable region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 150. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to TIGIT, the polynucleotide includes SEQ ID NO: 144 and SEQ ID NO: 149.

[0221] In some embodiments, the polynucleotide can encode a Fab that binds immunospecifically to TIGIT, wherein the Fab includes the amino acid sequence of SEQ ID NO: 158. The polynucleotide can encode a Fab that includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 158. The polynucleotide can encode a Fab that includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 158. In some embodiments, in a polynucleotide encoding a Fab that binds immunospecifically to TIGIT, the polynucleotide includes SEQ ID NO: 157.

[0222] A polynucleotide can encode any antibody disclosed herein, the antibody having the isotype IgG.

[0223] This specification discloses vectors comprising any of the nucleic acid molecules described herein, including isolated polypeptides or polynucleotides encoding antibodies. This specification also discloses host cells comprising any of the vectors disclosed herein. Suitable host cells include eukaryotic cells and prokaryotic cells.

[0224] This specification discloses a method for producing an isolated polypeptide or antibody, the method comprising culturing a host cell containing one of the vectors disclosed herein under conditions suitable for the expression of the isolated polypeptide or antibody, and purifying the isolated polypeptide or antibody.

[0225] In this specification, the formula I:Z-(XY n -Z r ) p An antibody is disclosed comprising an isolated polypeptide containing the amino acid sequence -X, where Z is a hinge region, X is a CH2 domain, Y is a linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, and r is an integer of 0 or 1.

[0226] The hinge (Z) region may contain the amino acid sequence of SEQ ID NO: 18. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. Hinge (Z) may contain the amino acid sequence of SEQ ID NO: 48. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. Hinge (Z) may contain the amino acid sequence of SEQ ID NO: 102. Hinge (Z) may contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 102. Hinge (Z) may contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 102.

[0227] 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.

[0228] In some embodiments, the antibody containing the isolated polypeptide may include one or more linkers (Y) containing the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. One or more linkers (Y) may include GS. One or more linkers (Y) may include GGS. One or more linkers (Y) may include SEQ ID NO: 14. One or more linkers (Y) may include SEQ ID NO: 38. One or more linkers may include SEQ ID NO: 43. One or more linkers (Y) may include sequences having at least 90% sequence identity with SEQ ID NO: 43. One or more linkers (Y) may include SEQ ID NO: 44. One or more linkers (Y) may include sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0229] An isolated polypeptide can contain one linker (Y) if n is 1. An isolated polypeptide can contain two linkers (Y) if n is 2. An isolated polypeptide can contain three linkers (Y) if n is 3. An isolated polypeptide can contain four linkers (Y) if n is 4. An isolated polypeptide can contain five linkers (Y) if n is 5.

[0230] In some embodiments, the antibody containing the isolated polypeptide has a CH2-linker region ((XY n ) p The isolated polypeptide may contain two or more regions including ((XY) such that p is 2 or more. n ) p ) may include three CH2-linker regions ((XY n ) p ) may include four CH2-linker regions ((XY n ) p) may include five CH2-linker regions ((XY n ) p ) can include

[0231] In some embodiments, the antibody containing the isolated polypeptide has a CH2-linker-hinge region ((XY n -Z r ) p The isolated polypeptide may contain two or more regions including ((XY) such that p is 2 or more. n -Z r ) p ) may contain three CH2-linker-hinge regions ((XY n -Z r ) p ) may contain four CH2-linker-hinge regions ((XY n -Z r ) p ) may contain five CH2-linker-hinge regions ((XY n -Z r ) p ) can include

[0232] In some embodiments, the antibody containing the isolated polypeptide is (XY n -Z r ) p It contains, essentially consists of, or is composed of, where 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, n is 1, r is 0 or 1, and p is 2.

[0233] In some embodiments, the antibody containing the isolated polypeptide is (XY n -Z r )p comprises, consists essentially of, or consists of, wherein Z (hinge) comprises the amino acid sequence of SEQ ID NO: 48, X (CH2 domain) comprises the amino acid sequence of SEQ ID NO: 49, Y (linker) comprises the amino acid sequence of SEQ ID NO: 44, n is 1, r is 0 or 1, and p is 2.

[0234] In some embodiments, the antibody comprising the isolated polypeptide is (X-Y n -Z r ) p comprises, consists essentially of, or consists of, wherein Z (hinge) comprises the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) comprises the amino acid sequence of SEQ ID NO: 104, Y (linker) comprises the amino acid sequence of SEQ ID NO: 44, n is 1, r is 0 or 1, and p is 2.

[0235] In some embodiments, the antibody comprising the isolated polypeptide is (X-Y n -Z r ) p comprises, consists essentially of, or consists of, wherein Z (hinge) comprises the amino acid sequence of SEQ ID NO: 102, X (CH2 domain) comprises the amino acid sequence of SEQ ID NO: 104, Y (linker) comprises the amino acid sequence of SEQ ID NO: 45, n is 1, r is 0 or 1, and p is 2.

[0236] In some embodiments, the antibody containing the isolated polypeptide contains 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 containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated polypeptide containing the CH3 domain contains SEQ ID NO: 52. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 52. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, linked via a hinge and optionally one or more linkers, may contain an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 52.

[0237] In some embodiments, the antibody containing the isolated polypeptide includes two additional CH2 domains at the N-terminus of the Fc domain, which is connected via a hinge and optionally one or more linkers, and contains the "LFLEPS" mutation in the lower hinge and CH2 domain. The "LFLEPS" mutation may include the following amino acid substitutions: L234F, L235E, and P331S. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, which is connected via a hinge and optionally one or more linkers, and containing the "LFLEPS" mutation in the lower hinge and CH2 domain includes SEQ ID NO: 54. The isolated polypeptide containing two additional CH2 domains at the N-terminus of the Fc domain, which is connected via a hinge and optionally one or more linkers, and containing the "LFLEPS" mutation in the lower hinge and CH2 domain may contain an amino acid sequence having at least 90% identity to SEQ ID NO: 54. An isolated polypeptide containing two additional CH2 domains at the N-terminal end of an Fc domain connected via a hinge and optionally one or more linkers, and containing the "LFLEPS" mutation within the lower hinge and CH2 domains, may contain an amino acid sequence having at least 95% identity to SEQ ID NO: 54.

[0238] In some embodiments, antibodies comprising the isolated peptides disclosed herein have at least a 1.5-fold reduction in background signal compared to the corresponding wild-type antibody. In some embodiments, the reduction in background signal is 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, or at least 5-fold compared to the corresponding wild-type antibody.

[0239] In some embodiments, an antibody comprising any isolated peptide disclosed herein has at least a 1.5-fold increase in sensitivity or signal enhancement compared to the corresponding wild-type antibody. In some embodiments, the increase in sensitivity is at least 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, at least 9-fold, at least 9.5-fold, or at least 10-fold compared to the corresponding wild-type antibody.

[0240] In this specification, an antibody that immunospecifically binds to parkin is disclosed, the antibody comprising, essentially consisting of, or consisting of a heavy chain (HC) and a light chain (LC), which pair to form a binding domain 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 6; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to parkin further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0241] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0242] 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: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain 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 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0243] The first hinge and the second hinge can 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 can each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0244] 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.

[0245] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0246] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0247] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0248] 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 also include 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 also include 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.

[0249] 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.

[0250] In this specification, an antibody that immunospecifically binds to OCT4 is disclosed, the antibody comprising, essentially consisting of, or consisting of a heavy chain (HC) and a light chain (LC), which pair to form a binding domain 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 31; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to OCT4 further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0251] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can each contain the amino acid sequence of SEQ ID NO: 19. The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0252] 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: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain 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 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0253] 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.

[0254] 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.

[0255] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0256] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0257] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0258] 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 also include 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 also include 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.

[0259] 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.

[0260] This specification discloses an antibody that immunospecifically binds to IDO1, the antibody comprising, essentially consisting of, or comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 68; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to IDO1 further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0261] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0262] 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: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain 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 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0263] The first hinge and the second hinge can 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 can each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0264] 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.

[0265] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0266] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0267] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0268] 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 also include 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 include a heavy chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 75 and a light chain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 77.

[0269] 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.

[0270] This specification discloses an antibody that immunospecifically binds to a FLAG-tag, the antibody comprising, essentially consisting of, or comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising: a heavy chain complementarity determination 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 165; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to the FLAG-tag further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0271] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0272] 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: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain 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 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0273] The first hinge and the second hinge can 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 can each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

[0274] 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.

[0275] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0276] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20.

[0277] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0278] 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 also include 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 also include 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.

[0279] In some embodiments, 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.

[0280] In this specification, a rabbit anti-mouse IgG secondary antibody (RAM) is disclosed, the antibody comprising, essentially consisting of, or consisting of a heavy chain (HC) and a light chain (LC), which pair to form a 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 an LCDR3 containing 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, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge. In some embodiments, the rabbit anti-mouse IgG secondary antibody further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0281] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0282] 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: 49. The first CH2 domain, the second CH2 domain, and the third CH2 domain 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 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49.

[0283] 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, and each may 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.

[0284] 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.

[0285] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0286] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 141. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 141. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 141.

[0287] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0288] 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 also include 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 also include 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.

[0289] In some embodiments, 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.

[0290] This specification discloses an antibody that immunospecifically binds to pERK1 / 2, the antibody comprising, essentially consisting of, or comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising: 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 87; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to pERK1 / 2 further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0291] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0292] The first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 102. In some embodiments, the first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 102. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 102. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 102.

[0293] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0294] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 106. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 106. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 106.

[0295] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0296] 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 also include 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 also include 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.

[0297] 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.

[0298] This specification discloses an antibody that immunospecifically binds to PSD-95, the antibody comprising, essentially consisting of, or comprising a heavy chain (HC) and a light chain (LC), which pair 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 an LCDR3 containing 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, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to PSD-95 further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0299] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104.

[0300] The first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 102. In some embodiments, the first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 102. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 102. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 102.

[0301] The first and second linkers may each contain the amino acid sequences of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. The first and second linkers may contain GS. The first and second linkers may contain GGS. The first and second linkers may contain SEQ ID NO: 14. The first and second linkers may contain SEQ ID NO: 38. The first and second linkers may contain SEQ ID NO: 43. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 43. The first and second linkers may contain SEQ ID NO: 44. The first and second linkers may contain sequences having at least 90% sequence identity with SEQ ID NO: 44.

[0302] In some embodiments, the antibody includes a CH3 domain containing the amino acid sequence of SEQ ID NO: 106. The antibody may include a CH3 domain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 106. The antibody may include a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 106.

[0303] In some embodiments, the antibody includes 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 also include 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 also include 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.

[0304] 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 also include 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 also include 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.

[0305] In some embodiments, 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.

[0306] This specification discloses an antibody that binds immunospecifically to TIGIT, the antibody comprising, essentially consisting of, or comprising a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising: a heavy chain complementarity determination 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 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being connected by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being connected by a second linker and an optional second hinge. In some embodiments, the antibody that immunospecifically binds to TIGIT further comprises a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0307] The first CH2 domain, the second CH2 domain, and the third CH2 domain can 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 can 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 can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19.

[0308] The first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first hinge and the second hinge can each contain the amino acid sequence of SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. The first hinge and the second hinge can each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18.

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

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

[0312] 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 also include 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 also include 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.

[0313] In some embodiments, 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.

[0314] 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 may be a fluorophore. The label may be a fluorescent protein. The label may be a phosphorescent dye. The label may be a tandem dye. The label may be a polymer dye. The label may be a particle. The label may be an electron transfer agent. The label may be a hapten. The label may be an enzyme. The label may be a radioisotope. The label may be a chromophore. The label may be an oligonucleotide-based fluorescent dye.

[0315] In some embodiments, the label is a fluorophore. The fluorophore may be pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, indacene, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), cyanine, carbocyanine, benzocyanine, carbostyryl, porphyrin, salicylic acid, anthranilic acid, azulene, perylene, pyridine, quinoline, borapoliazindacene, xanthene, rhodamine, siliconrhodamine, fluorescein, rhodol, rosamine, oxazine or benzoxazine, benzophenoxazine, carbazine, phenalenone, coumarin, furan or benzofuran, benzophenalenone, benzopyrillium, styryl, squaline, resolfin, 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 benzindole. The fluorophore can be oxazole. The fluorophore can be benzoxazole. The fluorophore can be thiazole. The fluorophore can be benzothiazole. The fluorophore can be indacene. The fluorophore can be 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). The fluorophore can be cyanine. The fluorophore can be carbocyanine. The fluorophore can be benzocyanine. The fluorophore can be carbostyryl. The fluorophore can be porphyrin. The fluorophore can be salicylic acid. The fluorophore can be anthranilic acid. The fluorophore can be azulene. The fluorophore can be perylene. The fluorophore may be pyridine. The fluorophore may be quinoline. The fluorophore may be borapolyazindacene. The fluorophore may be xanthene.The fluorophore can be rhodamine. The fluorophore can be silicon rhodamine. The fluorophore can be fluorescein. The fluorophore can be lordol. The fluorophore can be rosamine. The fluorophore can be oxazine. The fluorophore can be benzoxazine. The fluorophore can be benzophenoxazine. The fluorophore can be carbazine. The fluorophore can be phenalenone. The fluorophore can be coumarin. The fluorophore can be furan. The fluorophore can be benzofuran. The fluorophore can be benzophenalenone. The fluorophore can be benzopyrillium. The fluorophore can be styryl. The fluorophore can be squaline. The fluorophore can be resorphine. 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.

[0316] In some embodiments, the label is a particle. The particle may be a semiconductor nanocrystal.

[0317] In some embodiments, the label is a hapten. The hapten may be biotin or a biotin derivative. The hapten may be biotin. The hapten may be a biotin derivative.

[0318] In some embodiments, the label is an enzyme. The enzyme may be a peroxidase, phosphatase, glycosidase, or luciferase. The enzyme may be a peroxidase. The enzyme may be a phosphatase. The enzyme may be a glycosidase. The enzyme may be a luciferase.

[0319] Disclosed herein are nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that immunospecifically bind to parkin, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair to form a binding domain 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 6, 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0320] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the 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 containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 23.

[0321] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain contains SEQ ID NO: 62.

[0322] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0323] In some embodiments, the polynucleotides encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing 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, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0324] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0325] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0326] 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 can encode an antibody comprising 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 polynucleotide can encode an antibody comprising 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.

[0327] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 2 and SEQ ID NO: 11.

[0328] Disclosed herein are nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that immunospecifically bind to OCT4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair to form a binding domain 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 31, 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0329] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the 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 containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 23.

[0330] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain contains SEQ ID NO: 62.

[0331] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0332] In some embodiments, the polynucleotides encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing 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, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0333] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0334] 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 can encode an antibody comprising 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 polynucleotide can encode an antibody comprising 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.

[0335] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0336] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 27 and SEQ ID NO: 35.

[0337] Disclosed herein are nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding an antibody that immunospecifically binds to IDO1, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, and the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of a third CH2 domain.

[0338] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the 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 containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 178.

[0339] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain contains SEQ ID NO: 62.

[0340] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0341] In some embodiments, the polynucleotides encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing 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, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0342] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0343] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0344] 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 can encode an antibody comprising 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 polynucleotide can encode an antibody comprising 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.

[0345] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 75 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 77. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 75 and a light chain having at least 95% sequence identity to SEQ ID NO: 77. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 75 and SEQ ID NO: 77.

[0346] This specification discloses nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that immunospecifically bind to a FLAG-tag, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which, when paired, form a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 165, and a light chain complementarity determination region containing the amino acid sequence of SEQ ID NO: 168. A binding domain is formed comprising region 1 (LCDR1), LCDR2 containing the amino acid sequence of YAS, and LCDR3 containing the amino acid sequence of Sequence ID No. 169, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, and the second and third CH2 domains being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of the third CH2 domain.

[0347] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the 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 containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 23.

[0348] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain contains SEQ ID NO: 62.

[0349] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0350] In some embodiments, the polynucleotides encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing 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, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0351] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0352] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0353] 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 can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 162 and a light chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 167. The polynucleotide can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162 and a light chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 167.

[0354] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 171 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 173. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 171 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 173. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 170 and SEQ ID NO: 172.

[0355] This specification discloses nucleic acid molecules comprising, essentially composed of, or comprising polynucleotides encoding rabbit anti-mouse IgG secondary antibodies (RAM), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which, when paired, form a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 127, HCDR2 containing the amino acid sequence of SEQ ID NO: 128, HCDR3 containing the amino acid sequence of SEQ ID NO: 129, and a light chain complementarity-determining region containing the amino acid sequence of SEQ ID NO: 132. A binding domain is formed comprising 1(LCDR1), LCDR2 containing the amino acid sequence of GAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 133, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, and the second and third CH2 domains being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of the third CH2 domain.

[0356] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the 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 containing the amino acid sequence of SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 23.

[0357] In some embodiments, the polynucleotide encodes a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing the amino acid sequence of SEQ ID NO: 49. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49, and the polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49. In some embodiments, in the polynucleotide encoding the first CH2 domain, the second CH2 domain, and the third CH2 domain, the polynucleotide encoding each CH2 domain contains SEQ ID NO: 62.

[0358] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0359] In some embodiments, the polynucleotides encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. The polynucleotides can encode a first hinge and a second hinge, each containing 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, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0360] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0361] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0362] 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 can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 126 and a light chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 131. The polynucleotide can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126 and a light chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 131.

[0363] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 135 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 137. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 135 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 137. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 134 and SEQ ID NO: 136.

[0364] This specification discloses nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that immunospecifically bind to pERK1 / 2, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which, when paired, form 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 87, and a light chain complementarity-determining region 1 containing the amino acid sequence of SEQ ID NO: 91. A binding domain is formed comprising (LCDR1), LCDR2 containing the amino acid sequence of KVS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 92, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, and the second and third CH2 domains being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of the third CH2 domain.

[0365] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing 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 containing the amino acid sequence of SEQ ID NO: 104. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 103.

[0366] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 102. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 102. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 102. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 102. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 101.

[0367] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0368] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 106.

[0369] 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 can encode an antibody comprising 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 polynucleotide can encode an antibody comprising 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.

[0370] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 94 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 96. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 94 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 93 and SEQ ID NO: 95.

[0371] This specification discloses nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that immunospecifically bind to PSD-95, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which, when paired, comprise a heavy chain complementarity determination 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, and a light chain complementarity determination region containing the amino acid sequence of SEQ ID NO: 116. A binding domain is formed comprising 1(LCDR1), LCDR2 containing the amino acid sequence of DTS, and LCDR3 containing the amino acid sequence of Sequence ID No. 117, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, and the second and third CH2 domains being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of the third CH2 domain.

[0372] A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing 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 containing the amino acid sequence of SEQ ID NO: 104. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 104. A polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 104. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 103.

[0373] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 102. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 102. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 102. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 102. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 101.

[0374] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0375] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 106. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 106.

[0376] 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 can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110 and a light chain variable region having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 115. The polynucleotide can encode an antibody comprising a heavy chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 110 and a light chain variable region having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 115.

[0377] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 119 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 119 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 121. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 118 and SEQ ID NO: 120.

[0378] This specification discloses nucleic acid molecules comprising, essentially composed of, or consisting of polynucleotides encoding antibodies that bind immunospecifically to TIGIT, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which, when paired, comprise a heavy chain complementarity determination 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, and a light chain complementarity determination region containing the amino acid sequence of SEQ ID NO: 151. A binding domain is formed comprising 1(LCDR1), LCDR2 containing the amino acid sequence of SAS, and LCDR3 containing the amino acid sequence of Sequence ID No. 152, and the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first and second CH2 domains being linked by a first linker and an optional first hinge, and the second and third CH2 domains being linked by a second linker and an optional second hinge. In some embodiments, the polynucleotide further encodes a CH3 domain ligated to the C-terminus of the third CH2 domain.

[0379] The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing 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 containing the amino acid sequence of SEQ ID NO: 19. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19. The polynucleotide can encode a first CH2 domain, a second CH2 domain, and a third CH2 domain, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 19. In some embodiments, in a polynucleotide encoding a first CH2 domain, a second CH2 domain, and a third CH2 domain, the polynucleotide contains SEQ ID NO: 23.

[0380] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 22.

[0381] A polynucleotide can encode a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, a polynucleotide encodes a first hinge and a second hinge, each containing the amino acid sequence of SEQ ID NO: 48. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48. A polynucleotide can encode a first hinge and a second hinge, each containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48. In some embodiments, in a polynucleotide encoding a first hinge and a second hinge, each hinge-encoding polynucleotide contains SEQ ID NO: 51.

[0382] A polynucleotide can encode a first and second linker containing the amino acid sequence of GS, GGS, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 43, or SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing GS. The first and second linkers may contain GGS. A polynucleotide can encode a first and second linker containing SEQ ID NO: 14. A polynucleotide can encode a first and second linker containing SEQ ID NO: 38. A polynucleotide can encode a first and second linker containing SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 43. A polynucleotide can encode a first and second linker containing SEQ ID NO: 44. A polynucleotide can encode a first and second linker containing a sequence having at least 90% sequence identity with SEQ ID NO: 44.

[0383] In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an 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 to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes an antibody comprising a CH3 domain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20.

[0384] 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 can encode an antibody comprising 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 polynucleotide can encode an antibody comprising 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.

[0385] 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 can encode an antibody comprising a heavy chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 154 and a light chain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 156. The polynucleotide can encode an antibody comprising a heavy chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 154 and a light chain having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 156. In some embodiments, in a polynucleotide encoding a heavy chain and a light chain, the polynucleotide comprises SEQ ID NO: 153 and SEQ ID NO: 155.

[0386] This specification discloses a vector comprising one of the nucleic acid molecules described herein, including a polynucleotide encoding an antibody. This specification also discloses a host cell comprising one of the vectors disclosed herein. Suitable host cells include eukaryotic cells and prokaryotic cells.

[0387] The antibodies or polypeptides according to this disclosure may be produced by expression in, for example, a bacterial host (prokaryotic system) or a eukaryotic system such as yeast, fungi, insect cells, or mammalian cells, using any known and well-established expression system and recombinant cell culture technique. The antibodies or polypeptides according to this disclosure may be produced in transgenic organisms such as goats or plants. The antibodies or polypeptides according to this disclosure may also be produced by chemical synthesis.

[0388] This specification discloses a method for detecting the presence of a target protein in a sample, the method comprising, essentially comprising, or comprising: applying to a sample containing the target protein any of the isolated polypeptides disclosed herein or any of the antibodies disclosed herein; incubating the isolated polypeptide or antibody in the sample under conditions that enable the isolated polypeptide or antibody to bind to the target protein and form a bound isolated polypeptide or bound antibody; and determining the presence of the bound isolated polypeptide or bound antibody, thereby detecting the presence of the target protein.

[0389] This specification discloses a method for detecting the presence of a target protein in a sample, the method comprising, essentially comprising, or consisting of, applying one of the isolated polypeptides disclosed herein to a sample containing the target protein; incubating the isolated polypeptide in the sample under conditions that enable 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.

[0390] This specification discloses a method for detecting the presence of a target protein in a sample, the method comprising, essentially comprising, or consisting of, applying one of the antibodies disclosed herein to a sample containing the target protein; incubating the antibody in the sample under conditions that enable 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.

[0391] This specification discloses the use of any of the isolated polypeptides or antibodies disclosed herein for detecting the presence of a target protein in a sample. The use includes, essentially consists of, or may consist of, applying any of the isolated polypeptides or antibodies disclosed herein to a sample containing the target protein; incubating the isolated polypeptide or antibody in the sample under conditions that enable the isolated polypeptide or antibody to bind to the target protein and form a bound isolated polypeptide or bound antibody; and determining the presence of the bound isolated polypeptide or bound antibody, thereby detecting the presence of the target protein.

[0392] This specification discloses the use of any of the isolated polypeptides disclosed herein for detecting the presence of a target protein in a sample. The use includes, essentially, or may consist of, applying any of the isolated polypeptides disclosed herein to a sample containing the 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.

[0393] This specification discloses the use of any of the antibodies disclosed herein for detecting the presence of a target protein in a sample. The use includes, essentially, or may consist of, applying any of the antibodies disclosed herein to a sample containing the 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.

[0394] In some embodiments of the method or use, a smaller amount of the isolated polypeptide or antibody disclosed herein is required compared to the amount of wild-type isolated polypeptide or wild-type antibody that would be required to produce a detectable signal in an equivalent method. A smaller amount of the isolated polypeptide disclosed herein may be required compared to the amount of wild-type isolated peptide that would be required to produce a detectable signal in an equivalent method. A smaller amount of the antibody disclosed herein may be required compared to the amount of wild-type antibody that would be required to produce a detectable signal in an equivalent method. The isolated polypeptides and antibodies disclosed herein provide enhanced sensitivity at lower concentrations, enabling the generation of a strong signal at concentrations where wild-type isolated polypeptide or wild-type antibody would provide a weaker signal.

[0395] In some embodiments of the method or use, a smaller amount of sample (e.g., rare clinical sample) is used compared to an equivalent method using a wild-type isolated polypeptide or wild-type antibody, yet a larger signal is still generated.

[0396] In some embodiments of the method or use, lower concentrations of the target protein in a sample can be detected compared to equivalent methods using wild-type isolated polypeptides or wild-type antibodies.

[0397] In some embodiments of the method or use, the isolated polypeptides or antibodies disclosed herein have lower background binding to cells expressing Fc gamma receptor (FcγR) compared with wild-type isolated polypeptides or wild-type antibodies.

[0398] In some embodiments of the method or use, the target protein is OCT4, Parkin, IDO1, 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 Parkin. 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 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.

[0399] The target protein may be present in low abundance in the sample. Low abundance of the target means it is expressed or present at low levels, either below the detection limit or providing a very weak signal in standard immunoassays. Low abundance includes situations where the target protein cannot be detected or is difficult to detect in certain assays using wild-type antibodies.

[0400] In some embodiments of the method or use, the presence of a conjugated isolated polypeptide or a conjugated antibody is determined by applying a secondary antibody to the sample, the secondary antibody being labeled. In some embodiments of the method of use, the secondary antibody is the rabbit anti-mouse (RAM) antibody of the Disclosure.

[0401] 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 may be a fluorophore. The label may be a fluorescent protein. The label may be a phosphorescent dye. The label may be a tandem dye. The label may be a polymer dye. The label may be a particle. The label may be an electron transfer agent. The label may be a hapten. The label may be an enzyme. The label may be a radioisotope. The label may be a chromophore. The label may be an oligonucleotide-based fluorescent dye.

[0402] In some embodiments, the label is a fluorophore. The fluorophore may be pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, indacene, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), cyanine, carbocyanine, benzocyanine, carbostyryl, porphyrin, salicylic acid, anthranilic acid, azulene, perylene, pyridine, quinoline, borapoliazindacene, xanthene, rhodamine, siliconrhodamine, fluorescein, rhodol, rosamine, oxazine or benzoxazine, benzophenoxazine, carbazine, phenalenone, coumarin, furan or benzofuran, benzophenalenone, benzopyrillium, styryl, squaline, resolfin, 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 benzindole. The fluorophore can be oxazole. The fluorophore can be benzoxazole. The fluorophore can be thiazole. The fluorophore can be benzothiazole. The fluorophore can be indacene. The fluorophore can be 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). The fluorophore can be cyanine. The fluorophore can be carbocyanine. The fluorophore can be benzocyanine. The fluorophore can be carbostyryl. The fluorophore can be porphyrin. The fluorophore can be salicylic acid. The fluorophore can be anthranilic acid. The fluorophore can be azulene. The fluorophore can be perylene. The fluorophore may be pyridine. The fluorophore may be quinoline. The fluorophore may be borapolyazindacene. The fluorophore may be xanthene.The fluorophore can be rhodamine. The fluorophore can be silicon rhodamine. The fluorophore can be fluorescein. The fluorophore can be lordol. The fluorophore can be rosamine. The fluorophore can be oxazine. The fluorophore can be benzoxazine. The fluorophore can be benzophenoxazine. The fluorophore can be carbazine. The fluorophore can be phenalenone. The fluorophore can be coumarin. The fluorophore can be benzofuran. The fluorophore can be benzophenalenone. The fluorophore can be benzopyrillium. The fluorophore can be styryl. The fluorophore can be squaline. The fluorophore can be resorphine. 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.

[0403] In some embodiments, the label is a particle. The particle may be a semiconductor nanocrystal.

[0404] In some embodiments, the label is a hapten. The hapten may be biotin or a biotin derivative. The hapten may be biotin. The hapten may be a biotin derivative.

[0405] In some embodiments, the label is an enzyme. The enzyme may be a peroxidase, phosphatase, glycosidase, or luciferase. The enzyme may be a peroxidase. The enzyme may be a phosphatase. The enzyme may be a glycosidase. The enzyme may be a luciferase.

[0406] The method or use may further include adding an enzyme substrate selected from chromogenic substrates, fluorescence-generating substrates, and chemiluminescent substrates. The enzyme substrate may be a chromogenic substrate. The enzyme substrate may be a fluorescence-generating substrate. The enzyme substrate may be a chemiluminescent substrate.

[0407] The method or use may include detecting the labeled antibody using flow cytometry, immunohistochemistry, immunocytochemistry, or Western blotting. The labeled antibody can be detected using flow cytometry. The labeled antibody can be detected using immunohistochemistry. The labeled antibody can be detected using immunocytochemistry. The labeled antibody can be detected using Western blotting.

[0408] [Table 1-1] JPEG2026533932000003.jpg247160 JPEG2026533932000004.jpg109170

[0409] [Table 1-2] JPEG2026533932000006.jpg247159 JPEG2026533932000007.jpg68170

[0410] [Table 1-3] JPEG2026533932000009.jpg81170

[0411] [Table 2] JPEG2026533932000011.jpg186170

[0412] [Table 3-1] JPEG2026533932000013.jpg249162 JPEG2026533932000014.jpg208170

[0413] Table 3-2 JPEG2026533932000016.jpg249160 JPEG2026533932000017.jpg172170

[0414] Table 3-3 JPEG2026533932000019.jpg166170

[0415] Table 4 JPEG2026533932000021.jpg249160 JPEG2026533932000022.jpg20170

[0416] Table 5 JPEG2026533932000024.jpg25170

[0417] Table 6-1 JPEG2026533932000026.jpg134170

[0418] Table 6-2 JPEG2026533932000028.jpg149170

[0419] Table 7 JPEG2026533932000030.jpg157170

[0420] Table 8-1 JPEG2026533932000032.jpg250162 JPEG2026533932000033.jpg 10170

[0421] Table 8-2 JPEG2026533932000035.jpg21170

[0422] Table 9

[0423] Table 10 JPEG2026533932000038.jpg54170

[0424] Table 11 JPEG2026533932000040.jpg249162 JPEG2026533932000041.jpg65170

[0425] Table 12 JPEG2026533932000043.jpg124170

[0426] Table 13-1 JPEG2026533932000045.jpg250162 JPEG2026533932000046.jpg140170

[0427] Table 13-2 JPEG2026533932000048.jpg147170

[0428] Table 14-1 JPEG2026533932000050.jpg250170

[0429] Table 14-2 JPEG2026533932000052.jpg194170

[0430] Table 14-3 JPEG2026533932000054.jpg165170

[0431] Table 15 JPEG2026533932000056.jpg250163 JPEG2026533932000057.jpg25170

[0432] Table 16-1 JPEG2026533932000059.jpg138170

[0433] Table 16-2 JPEG2026533932000061.jpg248159 JPEG2026533932000062.jpg94170

[0434] Table 16-3 JPEG2026533932000064.jpg76170

[0435] Table 17 JPEG2026533932000066.jpg204170

[0436] Table 18-1 JPEG2026533932000068.jpg247159 JPEG2026533932000069.jpg47170

[0437] Table 18-2 JPEG2026533932000071.jpg249166

[0438] Table 18-3

[0439] Table 19 JPEG2026533932000074.jpg238153 JPEG2026533932000075.jpg86170

[0440] Table 20-1 JPEG2026533932000077.jpg192170

[0441] Table 20-2 JPEG2026533932000079.jpg248160 JPEG2026533932000080.jpg121170

[0442] Table 20-3 JPEG2026533932000082.jpg110170

[0443] Table 21 JPEG2026533932000084.jpg230170

[0444] Table 22 JPEG2026533932000086.jpg250161 JPEG2026533932000087.jpg20170

[0445] Table 23

[0446] Table 24-1 JPEG2026533932000090.jpg248160 JPEG2026533932000091.jpg 15170

[0447] Table 24-2 JPEG2026533932000093.jpg224170

[0448] Table 24-3 JPEG2026533932000095.jpg199170

[0449] Table 25 JPEG2026533932000097.jpg247161 JPEG2026533932000098.jpg66170

[0450] Table 26-1 JPEG2026533932000100.jpg177170

[0451] Table 26-2 JPEG2026533932000102.jpg249161 JPEG2026533932000103.jpg118170

[0452] [Table 26-3] JPEG2026533932000105.jpg90170 [Examples]

[0453] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0454] Example 1: Design, expression, and analysis of recombinant antibody 2TDCH2-LFLEPS design Materials and methods All materials were obtained from Thermo Fisher Scientific (Waltham, MA) unless otherwise specified.

[0455] Construction of overexpression constructs of anti-OCT4-2TdCH2-LFLEPS, anti-Parkin-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.

[0456] Heavy chain expression construct: A skeletal vector containing the rabbit 2TdCH2-LFLEPS antibody skeleton was synthesized in pCDNA3.4 with annotated restriction sites, as illustrated in Figure 2A.

[0457] To construct expression constructs of OCT4 and parkin antibody having a 2TdCH2-LFLEPS skeleton, the Fab regions of parental OCT4 (clone ID: 3H8L6) and parkin antibody (clone ID: 21H24L9) were cloned between restriction sites XBA1 and KAS1 in the aforementioned vector. Restriction analysis of the constructs confirmed the accuracy of the expression constructs (Figures 2C and 3C).

[0458] Light chain expression constructs: Constructs for the light chains in rabbit OCT4 (clone ID: 3H8L6) and parkin (clone ID: 21H24L9) are illustrated in Figures 2B and 3B. Similarly, other targets were cloned into rabbit 2TdCH2-LFLEPS or mouse 2TdCH2 skeletons, as shown in Table 27 below.

[0459] [Table 27]

[0460] Expression and production of recombinant antibody 2TdCH2-LFLEPS Heavy and light chains for the targets listed in Table 27 were transfected into Expi293F® cells in a 1:1 ratio using either the ExpiFectamine® 293 Transfection Kit (product number A14524, protocol manual: MAN0007814) or the ExpiFectamine® CHO Transfection (product number A29129, protocol manual: MAN0014337). Five or eight days after transfection, the supernatant was collected and purified across a POROS® MabCapture® A / POROS® MabCapture® G / CaptureSelect® IgG-Fc (Multispecies) affinity matrix column and mixed in PBS, pH 7.4. Monomer content was confirmed by passing the antibody through an HPLC column (BioSep® 5μm SEC-s3000 290Å, LC column 300×7.8mm, Ea).

[0461] Immunoassay: Immunocytochemistry: Immunofluorescence analysis was performed using 70% densely packed cells of the desired cell line. Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, and blocked with 2% BSA at room temperature for 1 hour. Cells were stained with appropriate antibodies. Imaging was performed at 60×, 40×, or 20× magnification. Nuclei were stained with ProLong® Diamond Antifade Mountant using DAPI (product number P36962) or Hoescht (product number H1399), and cytoskeletal F-actin staining was performed with rhodamine phalloidin (product number R415).

[0462] Western blotting: Known amounts of protein samples were subjected to electrophoresis using Novex® NuPAGE® 4-12% Bis-Tris gel (product number NP0322BOX), XCell SureLock® Electrophoresis System (product number EI0002), and Novex® Sharp Pre-Stained Protein Standard (product number LC5800). The separated proteins were then transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (product number IB21001) and detected by scanning with the indicated primary and secondary antibodies. Detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (product number WP20005) or Chemiluminescent SuperSignal® West Dura Extended Duration Substrate (product number 34075), and detected using iBright® FL 1500 (product number A44115).

[0463] Immunohistochemistry: To expose target proteins in tissue sections, heat-induced epitope recovery was performed on deparaffinized sections using eBioscience® IHC Antigen Retrieval Solution-High pH (10×) (product number 00-4956-58) diluted in 1× aqueous solution in a declocking chamber at 110°C for 15 minutes. After antigen recovery, sections were blocked at room temperature for 45 minutes with 2% normal goat serum in 1× PBS, and then explored overnight at 4°C in a humidified chamber with or without the indicated primary antibody at a 1:100 dilution in 0.1% normal goat serum. Detection was performed at room temperature for 1 hour using Goat anti-Rabbit IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor® Plus 488 (product number A32731) at a 1:2,000 dilution in 0.1% normal goat serum. Tissue autofluorescence was quenched using the ReadyProbes® Tissue Autofluorescence Quenching Kit (product number R37630). Nuclei were stained with DAPI (product number D1306), and sections were mounted using ProLong® Glass Antifade Mountant (product number P36984). Images were captured at 20× magnification using the EVOS® M7000 Imaging System (product number AMF7000) and deconvolved laterally.

[0464] Flow cytometry: Cells were fixed and permeabilized using either the Intracellular Fixation & Permeabilization Buffer Set (product number 88-8824-00) (for Perkin) or the Foxp3 / Transcription Factor Staining Buffer Set (product number 00-5523-00) (for OCT4), and then stained with the respective antibodies. The histogram for the protein of interest is shown as a solid line, and the histogram for the unstained population is shown as a dashed line (Figure 10 for Perkin, Figures 12A-12B for IDO1). Viable cells were used for analysis as determined by Fixable Viability Dye eFluor® 450 (product number 65-0863-14) or Violet fluorescent reactive dye (L34964 A). Flow cytometry data were obtained using the Attune® NxT Flow Cytometer (product number A29004).

[0465] Conjugation: For Alexa Fluor® Plus 555 dye conjugation, the antibody was dialyzed overnight at 4°C in bicarbonate buffer (0.05 M, pH=8.5). A 15-fold excess of the dye was mixed with the antibody and incubated at room temperature for 1 hour, protected from light. The excess dye was removed by passing the sample through a Dowex® column 1 × 4-400, 50% slurry (product no. 22858). The antibody conjugate was formulated at a final concentration of 0.1 mM in StabilZyme® Protein-Free Stabilizer (pH 6.3~7.3) with added EDTA.

[0466] For Alexa Fluor® Plus 647 dye conjugation, the antibody was dialyzed overnight at 4°C in bicarbonate buffer (0.05 M, pH=8.5). An 11-fold molar excess of the dye was mixed with the antibody and incubated at room temperature for 1 hour, protected from light. The excess dye was removed by passing the sample through a Dowex® column 1 × 4-400, 50% slurry (product no. 22858). The antibody conjugate was formulated at a final concentration of 0.1 mM in StabilZyme® Protein-Free Stabilizer (pH 6.3-7.3) supplemented with EDTA.

[0467] For SiteClick® conjugation of Alexa Fluor® 488 dye, the carbohydrate domain of the antibody was first modified, and azide conjugation was performed using the SiteClick® Antibody Azido Modification Kit (product number S10901). Subsequently, for antibody-labeled Alexa Fluor 647 (product number S10906), the sDIBO Alkyne-labeled (Alexa Fluor® 647-sDIBO) dye was conjugated to the azide-modified antibody using SiteClick® sDIBO Alkyne Kits. Excess dye was removed by passing the sample through Zeba® Dye and Biotin Removal Spin Columns and Filter Plates, 2 mL (product number A44299). The antibody conjugate was incorporated into 1× Tris pH 7.0 (or TBS) with 0.02% sodium azide added.

[0468] List of antibodies used in the immunoassay: All antibodies were obtained from Thermo Fisher Scientific unless otherwise specified. Parent Perkin recombinant rabbit monoclonal antibody (21H24L9) (product number 702785); Parent OCT4 recombinant rabbit monoclonal antibody (3H8L6) (product number 701756); Parent FLAG-tagged recombinant rabbit monoclonal antibody (8H8L17), (product number 701629); Parent IDO1 recombinant rabbit monoclonal antibody (7H8L17), (product number 702743); Parent TIGIT recombinant mouse monoclonal antibody (MBSA43), (product number 740010M); Parent PSD-95 recombinant mouse monoclonal antibody (7E3-1B8), (product number MA1-046); Goat anti-rabbit IgG (heavy chain); Superclonal® recombinant secondary antibody; Alexa Fluor® 647 (Product No. A27040); Goat anti-rabbit IgG(H+L) poly-HRP secondary antibody, HRP (Product No. 32260); Goat anti-rabbit IgG (heavy chain), Superclonal® recombinant secondary antibody, HRP (Product No. A27036); Goat anti-rabbit IgG(H+L) polyclonal secondary antibody, HRP (Catalog No. 31460); Donkey anti-rabbit IgG(H+L) secondary antibody, Alexa Fluor® Plus 488 (Product No. A32790); Donkey anti-rabbit IgG(H+L) secondary antibody, Alexa Fluor® Plus 488 (Product No. A32795); Goat anti-mouse IgG(H+L) Superclonal® secondary antibody (Product No. A28177).

[0469] List of reagents used in the immunoassay: All reagents were obtained from Thermo Fisher Scientific unless otherwise specified. Flow Cytometry Staining Buffer (product number 00-4222-26), IC Fixation Buffer (product number 00-8222-49), Foxp3 / Transcription Factor Staining Buffer Set (product number 00-5523-00), Fixable Viability Dye eFluor® 450 (product number 65-0863-14), Violet fluorescent reactive dye, for 405nm excitation (product number L34964 A), DAPI (product number D1306), Hoescht 33342, Trihydrochloride Trihydrate (product number H1399), ProLong® Glass Antifade Mountant (product number P36984).

[0470] siRNA: OCT4-specific siRNA (Silencer® select product numbers s10871, s10873), Parkin-specific siRNA (Silencer® select product numbers s530941, s530942).

[0471] result Production of rabbit and mouse recombinant antibodies with Fc-modified skeletons To evaluate the effect of the 2TdCH2 Fc manipulation skeleton, three rabbit antibodies were prepared: one in which the heavy chain (HC)Fab region (SEQ ID NO: 8) and light chain (LC)Fab region (SEQ ID NO: 11) were obtained from a Perkin rabbit recombinant antibody (clone ID: 21H24L9, product number 702785); another in which the HC Fab region (SEQ ID NO: 33) and LC Fab region (SEQ ID NO: 35) were obtained from an OCT4 rabbit recombinant antibody (clone ID: 3H8L6, product number 701756); and yet another in which the HC Fab region (SEQ ID NO: 78) and LC region (SEQ ID NO: 76) were obtained from an IDO1 rabbit recombinant antibody (clone ID: 7H8L17, product number 702743). The HC Fab region was then cloned into a vector containing either rabbit 2TdCH2 with the LFLEPS mutation (SEQ ID NO: 55) or rabbit 2TdCH2 without the LFLEPS mutation (SEQ ID NO: 53). Antibodies were also produced by cloning these Fabs into vectors containing either one additional Fc unit (1TdFc) containing nucleic acid with SEQ ID NO: 57 (encoding amino acid SEQ ID NO: 56), or two additional Fc units (2TdFc) containing nucleic acid with SEQ ID NO: 61 (encoding amino acid SEQ ID NO: 60). Schematic diagrams of the different operational designs are provided in Figures 1A to 1E. The yield, purity, and stability of these constructs were compared (Tables 28 and 29). Antibodies with the 2TdCH2 or 2TdCH2-LFLEPS backbone were comparable to the parental wild-type antibody in terms of yield, purity, and stability (Tables 28 and 29). In contrast, antibodies with the 1TdFc or 2TdFc backbone had poorer yield, lower monomer purity, and lower stability compared to the parental wild-type antibody (Tables 28 and 29).

[0472] To evaluate whether the manipulated rabbit Fc skeleton can benefit chimeric antibodies containing rabbit Fc, we prepared anti-TIGIT mouse-rabbit chimeric recombinant antibodies in which the HC Fab region (SEQ ID NO: 157) and LC region (SEQ ID NO: 155) were obtained from a mouse TIGIT antibody (clone ID: MBSA43, product number 16-9500-82). These antibodies were then cloned into vectors containing rabbit HC 2TdCH2 with the LFLEPS mutation (SEQ ID NO: 55) or rabbit HC 2TdCH2 without the LFLEPS mutation (SEQ ID NO: 53). A schematic diagram of the manipulation design is provided in Figure 1E. To evaluate the effect of the 2TdCH2+LFLEPS Fc manipulation scaffold on rabbit secondary antibodies, rabbit anti-mouse IgG (RAM) secondary antibodies were prepared by obtaining the HC Fab region (SEQ ID NO: 138) from a rabbit anti-mouse secondary antibody (clone ID: RAM35HC35LC37). These were then cloned into vectors containing rabbit 2TdCH2 with the LFLEPS mutation (SEQ ID NO: 55) or rabbit 2TdCH2 without the LFLEPS mutation (SEQ ID NO: 53). Schematic diagrams of the manipulation designs are provided in Figures 1D and 1E, respectively.

[0473] Additionally, the effect of the 2TdCH2 Fc design was investigated for mouse antibodies. The mouse IgG1 Fc domain lacks the amino acid that mediates interaction with FcγR1; therefore, the LFLEPS mutation does not provide further benefit to background reduction for this Fc isotype. Accordingly, mouse antibodies were prepared using either the HC Fab region (SEQ ID NO: 97) obtained from mouse pERK1 / 2 (clone ID: MILAN8R, product number 14-9109-82) or the HC Fab region (SEQ ID NO: 122) obtained from mouse PSD-95 hybridoma antibody (clone ID: 7E3-1B8, product number MA1-046), and cloned into vectors containing mouse IgG1 2TdCH2. A schematic diagram of the operational design is provided in Figure 1D.

[0474] [Table 28]

[0475] [Table 29]

[0476] Parkin and OCT4 antibodies with a 2TdCH2 skeleton demonstrate increased sensitivity in Western blotting and immunocytochemistry. The performance of the 2TdCH2-modified antibodies was first compared to that of the parent antibody. In Western blotting across different cell and / or tissue models, both the Perkin 2TdCH2 and OCT4 2TdCH2 antibodies demonstrated a significant sensitivity enhancement of approximately twofold compared to the wild-type parent antibody (Figure 5A, Figure 6A).

[0477] Table 30 illustrates the magnification changes normalized to WT for the rat brain blot in Figure 5A, and Table 31 illustrates the magnification changes normalized to WT for the PC12 blot in Figure 5A.

[0478] [Table 30]

[0479] [Table 31]

[0480] Table 32 illustrates the magnification change normalized to WT for the NTERA-2 blot in Figure 6A, and Table 33 illustrates the magnification change normalized to WT for the F9 blot in Figure 6A.

[0481] [Table 32]

[0482] [Table 33]

[0483] While the actual magnification changes may vary between experiments, cell / tissue models, and antibody concentrations used, the results were significant, as determined by independent Student's T-tests. Furthermore, there was no compromise on the specificity of the engineered antibodies, along with increased sensitivity, as demonstrated by the reduced signal in samples transfected with siRNA against Perkin or OCT4 and explored with the respective engineered antibodies (Figures 5B and 6C). Tests were conducted to determine if the benefits of the 2TdCH2 skeleton were limited to specific secondary antibodies. The 2TdCH2 skeleton was found to provide a significant and comparable increase in sensitivity regardless of the secondary antibody used, further highlighting the broad applicability of this design (Figures 7A and 7B). Next, tests were conducted to determine if mutating the residues involved in Fc receptor binding (LFLEPS) affected secondary antibody recognition. For both Parkin and OCT4, the sensitivity enhancement was comparable for both the 2TdCH2 and 2TdCH2-LFLEPS skeletons (Figure 5C, Figure 6B), demonstrating that the 2TdCH2-LFLEPS skeleton can be a universal skeleton for different applications. Next, the OCT4 2TdCH2 antibody was tested and compared to the parental OCT4 antibody in immunocytochemistry. It was found that the engineered OCT4 2TdCH2 antibody demonstrated an approximately 1.5-fold increase in signal-to-noise ratio, even when used at 1 / 8 the concentration of the parental antibody (Figure 8A). Furthermore, the specificity of the strong nuclear signal produced by the engineered antibody was confirmed using cells transfected with OCT4-specific siRNA (Figure 8B).

[0484] Parkin antibodies with the 2TdCH2 skeleton have an increased range of applicability compared to their parent antibodies. The parental rabbit recombinant Perkin antibody is sufficiently sensitive only in Western blotting. To test whether the engineered Perkin 2TdCH2-LFLEPS antibody is sufficiently sensitive for a wider range of applications, this antibody was tested in additional applications. In immunohistochemistry, the parental antibody was not sufficiently sensitive to be detectable in its application, but the engineered Perkin 2TdCH2-LFLEPS antibody was found to produce a strong signal and a staining pattern comparable to the benchmark rabbit polyclonal Perkin antibody (Thermo Fisher Scientific, catalog no. PA5-13399) (Figures 9A and 9B). In flow cytometry, the parental Perkin antibody showed only base-level staining, while the engineered antibody demonstrated an almost 40-fold increase in staining index (Figure 10). Table 34 shows the staining index for the antibodies shown in the flow cytometry analysis in Figure 10.

[0485] [Table 34]

[0486] Ido1 antibodies with the 2TdCH2+LFLEPS skeleton exhibit reduced background compared to their parent antibody. To test the effect of the LFLEPS mutation on the binding of rabbit antibodies to the FcR receptor, a system was used in which GFP-fused FcγR1 (CD64) was overexpressed in HEK293 cells. Immunofluorescence staining was performed using wild-type, engineered IDO1 with the LFLEPS mutation, and engineered IDO1 with the 2TdCH2+LFLEPS mutation. Since there is no endogenous expression of IDO1 in CD64-overexpressing HEK293 cells, this experiment purely tracks the binding of rabbit Fc to FcγR1, which represents background staining in immunoassays. As shown in Figure 11, there was significant background binding of the IDO1 antibody to cells in the case of wild-type and 2TdCH2 engineered antibodies. However, the introduction of the LFLEPS mutation in the context of either wild-type ("LFLEPS") or 2TdCH2 ("2TdCH2 LFLEPS") rabbit Fc dramatically eliminated this background binding. Similar results were observed in flow cytometry staining of CD64-overexpressing HEK293 cells using different IDO1 recombinant antibodies (Figures 12A-12B). As with the immunofluorescence experiment, since there is no endogenous expression of IDO1 in this model, the staining index (FcγRI binding by antibody) in Figure 12B shows background binding.

[0487] To evaluate the effect of signal enhancement associated with background reduction by the 2TdCH2+LFLEPS scaffold, CD64-GFP overexpressing HEK293 cells were subjected to interferon-gamma (IFNγ) treatment, which has been shown to increase endogenous IDO1 expression in these cells (Figures 13A-13B). In this model, anti-Ido1 antibody binding arises from both the antibody recognizing its target antigen Ido1 (signal) and arbitrary nonspecific binding of anti-Ido11 Fc to CD64 (background). Increased staining of anti-Ido1 wild-type antibody against CD64-transfected cells was observed compared to untransfected cells. Treatment of transfected cells with IFNγ did not result in any perceptible change in the mean intensity (signal / noise) of wild-type, LFLEPS, or 2TdCH2 anti-Ido1 antibodies (Figures 13A and 13B). (The mean circular intensity or mean intensity of the signal divided by noise is equal to signal / noise.) This is likely due to the low signal (wild-type and LFLEPS) and high background (wild-type and 2TdCH2) when using these antibodies. However, when these cells were stained with anti-IDO1 antibodies engineered with the 2TdCH2+LFLEPS scaffold, a sharp increase in the staining index (signal / noise) was observed compared to wild-type staining (2×) in treated cells and 2TdCH2+LFLEPS staining (approximately 3×) in untreated cells (Figures 13A and 13B). These results indicate that the engineer for increased sensitivity (2TdCH2), combined with the engineer for reduced background (LFLEPS mutation), provides a strong increase in signal relative to background.

[0488] TIGIT mouse-rabbit chimeric antibody with rabbit 2TdCH2+LFLEPS skeleton demonstrates increased sensitivity. To test the usefulness of rabbit 2TdCH2+LFLEPS as a universal rabbit skeleton with increased sensitivity in species-switching antibodies, a chimeric antibody was tested in which the Fab region of a mouse TIGIT IgG1...

Claims

1. Equation I: Z - (X - Y n -Z r ) p An isolated polypeptide comprising the amino acid sequence -X, wherein, Z is the hinge, X is the CH2 domain, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, An isolated polypeptide where r is an integer of 0 or 1.

2. Z contains an amino acid sequence that has at least 90% identity with SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO:

102. X contains an amino acid sequence that has at least 90% identity with SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, The isolated polypeptide according to claim 1, wherein r is an integer of 0 or 1.

3. The isolated polypeptide according to claim 1 or 2, wherein Y comprises an amino acid sequence having at least 90% identity with GS, GGS, 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. An isolated polypeptide according to any one of claims 1 to 3, wherein Z includes SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102, X includes SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y includes 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 more, p is an integer of 2 or more, and r is an integer of 0 or 1.

5. An 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. An 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. An 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 claims 1 to 7, wherein the isolated polypeptide is linked to a second polypeptide by one or more disulfide bonds, and the isolated polypeptide and the second polypeptide share the same amino acid sequence.

9. The isolated polypeptide according to any one of claims 1 to 8, 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 formed between the hinge of the isolated polypeptide and the hinge of the second polypeptide.

11. The isolated polypeptide according to any one of claims 1 to 10, 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 an isolated polypeptide according to any one of claims 1 to 12, ligated to a Fab domain.

14. The antibody according to 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 according to claim 13, wherein the Fab comprises a Fab heavy chain and a Fab light chain that pair to form a binding domain.

17. The antibody according to any one of claims 13 to 16, wherein the Fab binds immunospecifically to parkin, 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. Equation I: Z - (X - Y n -Z r ) p An antibody comprising a polypeptide containing the amino acid sequence -X, in the formula Z is the hinge, X is the CH2 domain, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, An antibody in which r is an integer of 0 or 1.

20. Z contains an amino acid sequence that has at least 90% identity with SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO:

102. X contains an amino acid sequence that has at least 90% identity with SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, The antibody according to claim 19, wherein r is an integer of 0 or 1.

21. The antibody according to claim 19 or 20, wherein Y comprises an amino acid sequence having at least 90% identity with GS, GGS, 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 includes SEQ ID NO: 18, SEQ ID NO: 48, or SEQ ID NO: 102, X includes SEQ ID NO: 19, SEQ ID NO: 49, or SEQ ID NO: 104, Y includes 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 more, p is an integer of 2 or more, 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 a second polypeptide by one or more disulfide bonds, and 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 according to claim 26, wherein the one or more disulfide bonds are formed 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, further comprising 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 antibody has at least a 1.5-fold reduction in background signal compared to the corresponding wild-type antibody that does not have the isolated polypeptide.

32. The antibody according to any one of claims 13 to 31, wherein the antibody has at least a 1.5-fold increase in sensitivity compared to the corresponding wild-type antibody that does not have the isolated polypeptide.

33. An isolated polypeptide according to any one of claims 1 to 12, further comprising a label, or an antibody according to any one of claims 13 to 32.

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

35. An antibody that immunospecifically binds to parkin, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair to form a binding domain comprising: a heavy chain complementarity determination 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; an HCDR3 containing the amino acid sequence of SEQ ID NO: 6; 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, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and 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 contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

37. The antibody according to claim 35 or 36, wherein the first hinge and the second hinge each contain 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 linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the 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 binds immunospecifically to OCT4, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 31, a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 37, an LCDR2 containing the amino acid sequence GAS, and an amino acid sequence containing the amino acid sequence of SEQ ID NO:

39. An antibody comprising an LCDR3 and a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

45. The antibody according to claim 43 or 44, wherein the first hinge and the second hinge each contain 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 linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 32 and a light chain variable region having the 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 binds immunospecifically to IDO1, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 68, 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 amino acid sequence of SEQ ID NO:

73. An antibody comprising an LCDR3, forming a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

53. The antibody according to claim 51 or 52, wherein the first hinge and the second hinge each contain 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 linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 65 and a light chain variable region having the 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 binds immunospecifically to a FLAG-tag, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 165, 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 amino acid sequence of SEQ ID NO: 169 An antibody comprising an LCDR3 containing an acid sequence, forming a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

61. The antibody according to claim 59 or 60, wherein the first hinge and the second hinge each contain 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 linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the 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 that is a rabbit anti-mouse IgG secondary antibody, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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 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 amino acid sequence of SEQ ID NO: 133 An antibody comprising an LCDR3 containing a sequence, forming a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:

49.

69. The antibody according to claim 67 or 68, wherein the first hinge and the second hinge each contain 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 linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having the 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 binds immunospecifically to pERK1 / 2, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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, an HCDR3 containing the amino acid sequence of SEQ ID NO: 87, a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 91, an LCDR2 containing the amino acid sequence of KVS, and an amino acid sequence of SEQ ID NO: 92 An antibody comprising an LCDR3 comprising a binding domain comprising a heavy chain further comprising a first CH2 domain, a second CH2 domain, and a third CH2 domain, wherein the first CH2 domain and the second CH2 domain are linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain are linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO:

104.

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

102.

78. The antibody according to any one of claims 75 to 77, wherein the first linker and the second linker each contain 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 the polypeptide sequence of SEQ ID NO: 83 and a light chain variable region having the 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 binds immunospecifically to PSD-95, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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 determination region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 116, an LCDR2 containing the amino acid sequence of DTS, and the amino acid sequence of SEQ ID NO: 117 An antibody comprising an LCDR3 containing a sequence, forming a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO:

104.

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

102.

86. The antibody according to any one of claims 83 to 85, wherein the first linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the 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 binds immunospecifically to TIGIT, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), which pair together to form a heavy chain complementarity determination 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 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 amino acid sequence of SEQ ID NO: 152 An antibody comprising an LCDR3 containing a sequence, forming a binding domain, wherein the heavy chain further comprises a first CH2 domain, a second CH2 domain, and a third CH2 domain, the first CH2 domain and the second CH2 domain being linked by a first linker and an optional first hinge, the second CH2 domain and the third CH2 domain being linked by a second linker and an optional second hinge, and the heavy chain further comprising 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 contain the amino acid sequence of SEQ ID NO:

19.

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

18.

94. The antibody according to any one of claims 91 to 93, wherein the first linker and the second linker 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.

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 the polypeptide sequence of SEQ ID NO: 145 and a light chain variable region having the 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 1.5-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 1.5-fold 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, wherein the method is Applying an isolated polypeptide according to any one of claims 1 to 12 or an antibody according to any one of claims 13 to 33 or 35 to 100 to a sample containing a target protein, The isolated polypeptide or antibody is incubated in the sample under conditions that enable the isolated polypeptide or antibody to bind to the target protein and form a bound isolated polypeptide or bound antibody. A method comprising determining the presence of the conjugated isolated polypeptide or the conjugated antibody, thereby detecting the presence of the target protein.

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

103. The method according to claim 101 or 102, wherein the target protein is OCT4, parkin, 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 a low amount.

105. The method according to any one of claims 101 to 104, wherein the presence of the conjugated isolated polypeptide or the conjugated antibody is determined by applying a secondary antibody to the sample, and 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 according to claim 105 or 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 benzindole, oxazole or benzoxazole, thiazole or benzothiazole, indacene, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), cyanine, carbocyanine, benzocyanine, carbostyryl, porphyrin, salicylic acid, anthranilic acid, azulene, perylene, pyridine, quinoline, borapoliazindacene, xanthene, rhodamine, silicone rhodamine, fluorescein, rhodol, rosamine, oxazine or benzoxazine, benzophenoxazine, carbazine, phenalenone, coumarin, benzofuran, benzophenalenone, benzopyrillium, styryl, squaline, resolphin, anthraquinone, or quinazolinone, or a conjugated polymer dye.

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

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

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

112. The method according to claim 111, further comprising adding an enzyme substrate selected from a chromogenic substrate, a fluorescence-generating substrate, and a chemiluminescent substrate.

113. The method according to any one of claims 106 to 112, wherein 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, said method comprising introducing into the heavy chain of said antibody a polypeptide comprising the amino acid sequence of Formula I: Z-(X-Y n -Z r ) p -X, wherein in the formula, Z is the hinge, X is the CH2 domain, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, r is an integer of 0 or 1, A method wherein the sensitivity of the antibody is increased by at least 1.5 times compared to the corresponding wild-type antibody.

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

117. The method according to claim 115 or 116, wherein X includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, at least 90% identical to SEQ ID NO: 49, or at least 90% identical to SEQ ID NO:

104.

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

102.

119. The method according to any one of claims 115 to 118, wherein Y comprises an amino acid sequence that is at least 90% identical to 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, wherein the method involves adding the formula I:Z-(X-Y) to the heavy chain of the antibody. n -Z r ) p This includes introducing a polypeptide containing the amino acid sequence of -X, in the formula Z is the hinge, X is the CH2 domain, Y is the linker, n is an integer greater than or equal to 1, p is an integer greater than or equal to 2, r is an integer of 0 or 1, A method wherein the background signal of the antibody is reduced by at least 1.5 times compared to the corresponding wild-type antibody.

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

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