Multivalent linkers for antibody labeling

Abstract

The present disclosure relates to tagging targets (e.g., primary antibodies) using multivalent linkers. The present disclosure provides methods of labeling antibodies with reporter / dye / enzyme via the multivalent linkers. The present disclosure further provides methods of using the multivalent linkers to label multiple primary antibodies of the same species in the same experiment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 396,519, filed on August 9, 2022, the contents of which are incorporated herein by reference in their entirety. Reference to an electronic sequence listing

[0002] The contents of the electronic sequence listing (PTGI_001_01WO_SeqList_ST26.xml; size: 535,676 bytes; creation date: August 8, 2023) are incorporated herein by reference in its entirety. Background Art

[0003] Immunodetection assays utilize the antigen binding ability of antibodies to detect and quantify the epitope of interest. These assays typically use a dual primary and secondary antibody system, where a specific primary antibody is developed to bind the target of interest, and a labeled secondary antibody is designed to generally target a conserved region within the primary antibody. In some cases, the primary antibody is directly labeled. However, current labeling and immunoassay technologies have several disadvantages, including inconsistent labeling, damage to epitope sites, antibody cross-reactivity, etc.

[0004] To overcome these challenges, additional techniques are needed. Summary of the invention

[0005] The present disclosure solves the above problems by creating and utilizing artificial multivalent linkers that can bind and label primary antibodies with high affinity.

[0006] Thus, in some embodiments, the present disclosure teaches a multivalent linker that specifically binds to a target antigen, the multivalent linker comprising: a) a plurality of peptide binding arms, each binding arm capable of binding to an epitope in the same target antigen; and b) a linker segment operably covalently linked to the plurality of peptide binding arms. In some embodiments, the multivalent linker specifically binds to a target antigen. In some embodiments, the target antigen is a constant domain of an antibody. In some embodiments, the protein is expressed at a rate of less than or equal to 1.0×10 -4 K off (s -1 ) rate of binding to antibody.

[0007] In some embodiments, the present disclosure provides a method for detecting two or more target antigens in a sample, the method comprising contacting the sample with a first antibody specific for a first target antigen and a second antibody specific for a second target antigen. In some embodiments, the first antibody and the second antibody are linked or conjugated to a first multivalent linker and a second multivalent linker, respectively. In some embodiments, each multivalent linker is less than or equal to 1.0×10 -4 K off(s -1 ) rate specifically binds to the constant region of the first antibody or the second antibody. In some embodiments, each multivalent linker is attached to a reporter. In some embodiments, the reporters are not the same.

[0008] In one aspect, the present disclosure provides a multivalent linker that specifically binds to a target antigen unit, the multivalent linker comprising: a) a plurality of peptide binding arms, each binding arm being capable of binding to an epitope in the same target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

[0009] In one aspect, the present disclosure provides a molecular complex comprising: (i) a single target antigen unit; and (ii) a multivalent linker, the multivalent linker comprising: a) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

[0010] In some embodiments, the multivalent linker is less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate to the target antigen unit. In some embodiments, the multivalent linker is bound to the target antigen unit at a rate of less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate of dissociation from a single target antigen unit. In some embodiments, k off (s -1 ) rates were determined by biolayer interferometry (BLI).

[0011] In some embodiments, the target antigen unit comprises the constant region of an antibody. In some embodiments, the target antigen unit comprises the Fc region of an antibody. In some embodiments, the epitope is located on the CH2 domain, CH3 domain and / or CH4 domain of the constant region or the Fc region. In some embodiments, one or more epitopes of the peptide binding arm are located on the CH1 domain or CL domain of the target antigen unit.

[0012] In some embodiments, the target antigen unit is an antibody, F(ab')2, Fab2, Fab3 or IgNAR. In some embodiments, the antibody is IgG. In some embodiments, the IgG antibody is IgG1, IgG2, IgG3 or IgG4 subclass, optionally wherein the IgG antibody is a human antibody. In some embodiments, the IgG antibody is IgG1, IgG2a, IgG2b, IgG2c or IgG3 subclass; optionally wherein the IgG antibody is a mouse antibody. In some embodiments, the IgG antibody is IgG1, IgG2a, IgG2b or IgG2c subclass; optionally wherein the IgG antibody is a rat antibody. In some embodiments, the antibody is IgM. In some embodiments, the antibody is a heavy chain antibody. In some embodiments, the antibody is a guinea pig antibody, a mouse antibody, a rat antibody, a chicken antibody (e.g., IgY), a donkey antibody, a rabbit antibody, a human antibody, a goat antibody, a pig antibody, a horse antibody or a cow antibody.

[0013] In some embodiments, at least one of the peptide binding arms has cross-reactivity and can bind to antibodies from two or more species non-simultaneously; optionally, wherein the two or more species are selected from humans, mice, rats and rabbits. In some embodiments, at least one of the peptide binding arms has cross-reactivity and can bind to antibodies from rabbits and humans non-simultaneously.

[0014] In some embodiments, each peptide binding arm is specific for a different epitope of the same target antigen unit. In some embodiments, each peptide binding arm is specific for the same epitope, and wherein the target antigen unit comprises a plurality of the same epitopes.

[0015] In some embodiments, the multiple peptide binding arms are capable of binding to the same target antigen unit. In some embodiments, the multiple peptide binding arms do not bind to more than one target antigen unit.

[0016] In some embodiments, the multivalent linker is bivalent and comprises two peptide binding arms.

[0017] In some embodiments, less than 5%, 4%, 3%, 2% or 1% of the multiple peptide binding arms are cross-linked with different target antigen units. In some embodiments, the multiple peptide binding arms are separated by a sufficiently long distance factor to prevent the peptide binding arms from being cross-linked with more than one target antigen unit.

[0018] In some embodiments, the linker segment comprises a peptide. In some embodiments, the linker segment comprises between 5-50 amino acids. In some embodiments, the linker segment comprises between 10-40 amino acids. In some embodiments, the linker segment comprises between 20-30 amino acids. In some embodiments, the linker segment comprises about 25 amino acids, about 30 amino acids, or about 35 amino acids.

[0019] In some embodiments, in the extended conformation, the length of the linker segment is In some embodiments, in the extended conformation, the length of the linker segment is about In some embodiments, in the extended conformation, the length of the linker segment is about In some embodiments, in the extended conformation, the length of the linker segment is about between.

[0020] In some embodiments, the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -5 In some embodiments, the multivalent linker is less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate of dissociation from a single target antigen unit.

[0021] In some embodiments, the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -6 In some embodiments, the multivalent linker is less than or equal to 1.0×10 -6 The apparent k off (s -1 ) rate of dissociation from a single target antigen unit.

[0022] In some embodiments, the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -7 In some embodiments, the multivalent linker is less than or equal to 1.0×10 -7 The apparent k off (s -1 ) rate of dissociation from a single target antigen unit.

[0023] In some embodiments, the linker segment comprises one (G4S) unit. In some embodiments, the linker segment comprises more than 2 (G4S) units. In some embodiments, the linker segment comprises more than 3 (G4S) units. In some embodiments, the linker segment comprises more than 4 (G4S) units. In some embodiments, the linker segment comprises more than 5 (G4S) units. In some embodiments, the linker segment comprises more than 6 (G4S) units. In some embodiments, the linker segment comprises at most 4, at most 5, at most 6, at most 7, at most 8, or at most 9 (G4S) units.

[0024] In some embodiments, the linker segment comprises the amino acid sequence of GSTSGSGKSSEGKGEGSTSGSGKSG (SEQ ID NO: 495).

[0025] In some embodiments, at least 20%-25% of the amino acids in the peptide of the linker segment are glycine. In some embodiments, between 60%-90% of the amino acids in the peptide of the linker segment are glycine. In some embodiments, between 10%-30% of the amino acids in the peptide of the linker segment are serine or threonine; more preferably, serine. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), aspartic acid (D), lysine (K) and arginine (R). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are glycine, alanine, serine and threonine. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine, serine, and threonine. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine and serine. In some embodiments, the ratio of (i) glycine to (ii) serine and / or threonine in the linker segment is about 4:1.

[0026] In some embodiments, the multivalent linker comprises at least one portion for conjugation with a heterologous molecule. In some embodiments, the portion for conjugation is cysteine. In some embodiments, the portion for conjugation is lysine. In some embodiments, the portion for conjugation comprises biotin or streptavidin. In some embodiments, the portion for conjugation comprises a functional group for conjugation by click chemistry. In some embodiments, the functional group includes a dibenzocyclooctyne group (DBCO), an azide, a tetrazine and / or a trans-cyclooctene (TCO).

[0027] In some embodiments, the linker segment comprises the moiety for conjugation. In some embodiments, the peptide in the linker segment comprises the moiety for conjugation.

[0028] In some embodiments, the heterologous molecule is a reporter, an oligonucleotide, a moiety functionalized for click chemistry, or an effector. In some embodiments, the multivalent linker comprises at least one attached reporter, an oligonucleotide, a moiety functionalized for click chemistry, or an effector. In some embodiments, the reporter is a fluorescent reporter. In some embodiments, the fluorescent reporter is a fluorescein dye, a rhodamine dye, two or more fluorescent dyes that can work in collaboration with each other, or a protein that exhibits fluorescence. In some embodiments, the fluorescent reporter is a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, a red fluorescent protein, mCherry, tdTomato, mStrawberry, mTangerine, and / or dsRed. In some embodiments, the reporter is an enzyme reporter. In some embodiments, the enzyme reporter is horseradish peroxidase, cathepsin, matrix metalloproteinase, peptidase, carboxypeptidase, glycosidase, lipase, phospholipase, phosphatase, phosphodiesterase, sulfatase, reductase, bacterial enzyme, biotin ligase, DNA transposase or nuclease. In some embodiments, the DNA transposase is Tn5 transposase. In some embodiments, the nuclease is micrococcal nuclease. In some embodiments, the effector is a magnetic effector. In some embodiments, the magnetic reporter is Gd(III), Dy(III), Fe(III), and Mn(II), DTPA, DOTA, DO3A, 2-benzyl-DOTA, α-(2-phenylethyl)1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA or 6,6″-bis[N,N,N″,N″-tetrakis(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

[0029] In some embodiments, the apparent K of the multivalent linker to the target antigen unit is D Less than 10,000 pM, less than 1,000 pM, less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 1 pM. In some embodiments, the apparent K of the multivalent linker to the target antigen unit is less than 10,000 pM, less than 1,000 pM, less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 1 pM. D 1 to 10 pM, 10 to 50 pM, 50 to 100 pM, 100 to 500 pM, or 500 to 1,000 pM. In some embodiments, the apparent K of the multivalent linker to the target antigen unit is 1 to 10 pM, 10 to 50 pM, 50 to 100 pM, 100 to 500 pM, or 500 to 1, D Less than about 50 pM. In some embodiments, the apparent K of the multivalent linker to the target antigen unit is D Less than about 25 pM. In some embodiments, the apparent K of the multivalent linker to the target antigen unit is D Less than about 10 pM.

[0030] In some embodiments, the plurality of peptide-binding arms comprises a peptide-binding arm comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID No: 1-494. In some embodiments, the plurality of peptide-binding arms comprises a peptide-binding arm comprising a sequence selected from the group comprising SEQ ID No 1-494.

[0031] In some embodiments, a multivalent linker or molecular complex of the present disclosure comprises more than one linker segment.

[0032] In some embodiments, the multivalent linker or molecular complex of the present disclosure comprises the structure: (peptide binding arm)-linker segment-(peptide binding arm).

[0033] In some embodiments, the multivalent linker or molecular complex of the present disclosure comprises the structure: (peptide binding arm)-linker segment-(peptide binding arm)-linker segment-(peptide binding arm).

[0034] In some embodiments, the peptide binding arm is a VHH of a camelid heavy chain antibody. In some embodiments, the peptide binding arm is a VH of an immunoglobulin. In some embodiments, the peptide binding arm comprises or consists of an immunoglobulin domain.

[0035] In some embodiments, the plurality of peptide binding arms are covalently linked to the linker segment. In some embodiments, the plurality of peptide binding arms and the linker segment form a continuous polypeptide.

[0036] In some embodiments, the plurality of peptide binding arms and the linker segment are operably linked by translational fusion. In some embodiments, the plurality of peptide binding arms, the linker segment, and the reporter or effector are each operably linked by translational fusion.

[0037] In some embodiments, each of the binding arms binds to an epitope of the target antigen unit. In some embodiments, the peptide binding arm non-covalently binds to the epitope.

[0038] In one aspect, the disclosure provides a composition comprising a multivalent linker of the disclosure or a molecular complex of the disclosure.

[0039] In some embodiments, the composition comprises a buffer.

[0040] In one aspect, the disclosure provides a composition comprising two or more different multivalent linkers of the disclosure or two or more different molecular complexes of the disclosure, wherein each of the multivalent linkers is linked to a different reporter.

[0041] In some embodiments, the target antigen unit comprises a binding domain that is capable of binding to a test antigen after the multivalent linker is bound to the target antigen unit.

[0042] In some embodiments, the composition further comprises a bait molecule comprising an epitope of the peptide binding arm or the plurality of peptide binding arms but not comprising the binding domain capable of binding to the test antigen.

[0043] In some embodiments, a cryoprotectant selected from glycerol, ethylene glycol and dimethyl sulfoxide (DMSO) is included. In some embodiments, the cryoprotectant is glycerol, and wherein the concentration of the glycerol is up to 50% by volume. In some embodiments, the glycerol concentration is less than 30% or less than 15% by volume. In some embodiments, the glycerol concentration is not less than 5% or not less than 10% by volume.

[0044] In one aspect, the present disclosure provides a method for detecting a test antigen in a sample, the method comprising the steps of: A) contacting the sample with a multivalent linker of the present disclosure and a binding agent capable of specifically binding to the test antigen, and B) removing unbound binder and multivalent linker, and C) measuring the presence of the multivalent linker in the sample; wherein the binding agent comprises the target antigen unit, and wherein the binding agent specifically binds to the test antigen.

[0045] In one aspect, the present disclosure provides a method for detecting two or more test antigens in a sample, the method comprising contacting the sample with a first binding agent specific for a first test antigen and a second binding agent specific for a second test antigen, wherein the first binding agent and the second binding agent are each bound to a first multivalent linker and a second multivalent linker, respectively, wherein the first multivalent linker and / or the second multivalent linker are multivalent linkers of the present disclosure, and wherein each multivalent linker is attached to a reporter, wherein the reporters are not the same.

[0046] In some embodiments, each multivalent linker is present in an amount less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate specifically binds to the constant region of the first binding agent or the second binding agent.

[0047] In some embodiments, the first binding agent and the second binding agent are each non-covalently bound to a first multivalent linker and a second multivalent linker, respectively. In some embodiments, the first binding agent and the second binding agent are each connected or conjugated to a first multivalent linker and a second multivalent linker, respectively.

[0048] In some embodiments, less than 5%, 4%, 3%, 2%, or 1% of the multivalent linkers are bound to two or more of the binding agents. In some embodiments, less than 5%, 4%, 3%, 2%, or 1% of the first multivalent linkers are bound to the second binding agent, and wherein less than 5%, 4%, 3%, 2%, or 1% of the second multivalent linkers are bound to the first binding agent.

[0049] In one aspect, the present disclosure provides a method for detecting one or more test antigens in a sample, the method comprising contacting the sample with one or more molecular complexes of the present disclosure, wherein the single target antigen unit in each of the molecular complexes is contained in a binding agent, and wherein the binding agent is capable of specifically binding to the test antigen.

[0050] In some embodiments, the methods are used to detect two or more different test antigens using two or more of the molecular complexes, wherein the binding agent of each of the molecular complexes is capable of specifically binding to one of the test antigens.

[0051] In some embodiments, the binding agent is an antibody or comprises an antigen-binding fragment thereof. In some embodiments, the first antibody and the second antibody are antibodies of the same species. In some embodiments, the first antibody and the second antibody are rabbit IgG antibodies. In some embodiments, the first antibody and the second antibody are mouse IgG antibodies. In some embodiments, the first antibody and the second antibody are rat IgG antibodies. In some embodiments, the first antibody and the second antibody are human IgG antibodies.

[0052] In some embodiments, the first multivalent linker is incubated with the first binding agent prior to contacting the sample with the first binding agent. In some embodiments, the second multivalent linker is incubated with the second binding agent prior to contacting the sample with the second binding agent. In some embodiments, the first binding agent is incubated with the first multivalent linker at a molar ratio of about 1:2.5. In some embodiments, the second binding agent is incubated with the second multivalent linker at a molar ratio of about 1:2.5.

[0053] In some embodiments, the stock concentration of the first binding agent is at least 0.001 g / l. In some embodiments, the stock concentration of the second binding agent is at least 0.001 g / l.

[0054] In some embodiments, the concentration of glycerol in the solution containing the first binding agent and / or the second binding agent is between 0-50% by volume. In some embodiments, the concentration of glycerol is less than 30% by volume, or less than 15% by volume. In some embodiments, the concentration of glycerol is not less than 5% by volume, or not less than 10% by volume.

[0055] In some embodiments, unbound multivalent linkers are quenched by adding a bait molecule that comprises the epitope of the peptide binding arm but does not bind to one or more of the test antigens.

[0056] In some embodiments, unbound multivalent linkers are removed from the multivalent linker-binder complex.

[0057] In some embodiments, unbound multivalent linkers are removed by ultrafiltration.

[0058] In some embodiments, the unbound multivalent linker is removed by bead depletion.

[0059] In some embodiments, unbound multivalent linkers are removed by adding nonspecific polyclonal IgG or fragments thereof.

[0060] In some embodiments, unbound multivalent linkers are removed by adding nonspecific monoclonal IgG or fragments thereof.

[0061] In some embodiments, the first multivalent linker and the first binding agent are incubated for about 30 minutes. In some embodiments, the first multivalent linker and the first binding agent are incubated for less than 10 minutes. In some embodiments, the second multivalent linker and the second binding agent are incubated for about 30 minutes. In some embodiments, the second multivalent linker and the second binding agent are incubated for less than 10 minutes.

[0062] In some embodiments, the method is used for Western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, immunohistochemistry, flow cytometry, fluorescence-assisted cell sorting (FACS), antibody screening (e.g., using hybridomas), spatial genomic analysis or mass spectrometry. In some embodiments, the method is used for cyclic immunofluorescence detection.

[0063] In one aspect, the present disclosure provides a molecular complex comprising: (a) a single target antigen unit, the single target antigen unit comprising: i) a constant region or Fc region of an antibody, wherein the constant region or Fc region is selected from: 1) Human IgG1, IgG2, IgG3 or IgG4; 2) mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3; 3) Rat IgG1, IgG2a, IgG2b or IgG2c; or 4) rabbit IgG; and (b) a multivalent linker, the multivalent linker comprising: i) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit, wherein said peptide binding arm is a VHH of a camelid heavy chain antibody; and ii) at least one peptide linker segment operably covalently linked to the plurality of peptide binding arms, wherein the peptide linker segment is between 10 and 40 amino acids in length, and wherein at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K) and arginine (R).

[0064] In some embodiments, each of the peptide binding arms of the multivalent linker is non-covalently linked to a CH2 domain, a CH3 domain, and / or a CH4 domain of a constant region or an Fc region of the antibody. In some embodiments, the linker segment comprises one (G4S) unit. In some embodiments, the linker segment comprises more than 3 (G4S) units. In some embodiments, the multiple peptide binding arms and the peptide linker segment are operably connected by translational fusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The patent or application file contains at least one drawing executed in color.

[0066] Figure 1 Results of biolayer interferometry are shown, depicting a comparison of the affinity of monovalent and multivalent linkers at different concentrations over time. In this case, a divalent multivalent linker according to the present disclosure at a concentration of 1.25 nM has a higher apparent affinity than a monovalent linker at 20 nM. A divalent multivalent linker at 20 nM is shown for comparison.

[0067] Figure 2 Results of biolayer interferometry are shown, depicting dissociation of the multivalent linker at concentrations of 0.6, 1.2, and 2.5 nM. Multivalent linker binding remained robust with little signal loss over the entire testing period.

[0068] Figure 3 Describe the result of immunofluorescence (IF) labeling experiment, this experiment compares the relative labeling signal intensity and leakage of various multivalent joints with different joint segment lengths and contrast monovalent joints.The multivalent joint of the present disclosure is less than the signal leakage suffered by the contrast monovalent joint.The multivalent joint with shorter (G4S) 3 (SEQ ID NO:527) joint segment shows certain signal leakage, but its level is lower than the monovalent control.The multivalent joint with longer (G4S) 4 (SEQ ID NO:528) and (G4S) 5 (SEQ ID NO:529) joint segment length does not show leakage substantially.The more detailed description of this experiment is provided in Example 6.

[0069] Figure 4A-4F Depicted are the results of multiplexed Western blot experiments using multivalent linkers of the present disclosure multiplexed with multiple primary antibodies from the same species. Figure 4A and Figure 4C is a scan of a Western blot treated with anti-Cox and anti-TDP primary antibodies of the same species labeled with two multivalent linkers comprising a reporter molecule detectable at 488 channels or a reporter molecule detectable at 647 channels, respectively. Figure 4B and Figure 4Dare controls for single, non-multiplexed western blots using the same anti-Cox primary antibody labeled with a multivalent linker containing a reporter molecule detectable at channel 488 or the anti-TDP primary antibody labeled with a multivalent linker containing a reporter molecule detectable at channel 647. Densitometry results for each western blot are presented in Figure 4E and Figure 4F The multivalent linkers of the present disclosure allow multiple assays to be performed using antibodies from the same species without signal leakage. A more detailed description of this experiment is provided in Example 7.

[0070] Figure 5 The crystal structure of the Fc fragment of mouse IgG1 antibody is shown (PDB ID 1igy). Distances are shown as distances between equivalent symmetric positions (dashed lines), used as a surrogate for distances between potential epitopes of the multivalent linkers of the present disclosure. Euclidean distances are given by express The epitope distances measured in the model correlate with empirical evidence demonstrating the consequences of various linker segment lengths in the multivalent linkers claimed in the present application. A more detailed description of the model and the measurements performed is provided in Example 10.

[0071] Fig. 6A Flow cytometry scatter plots of fluorescence activated single cell sorting (FACS) experiments using primary antibodies labeled with multivalent linkers of the present disclosure. Experiments were performed using different concentrations of glycerol (0-25%) to demonstrate the functionality of the multivalent linker over a wide range of glycerol concentrations. A more detailed description of the model and the measurements performed is provided in Example 8. Figure 6B Shown are the results of ELISA using GFP as antigen, anti-GFP primary antibody and HRP multivalent linker.

[0072] Figure 7 Depicts the results of a simulation evaluating the off The time required for 2% or 5% dissociation of a characteristic epitope binding molecule occurs. In some embodiments, the multivalent linker of the present disclosure is designed to have a low K off The values ​​show strong binding.

[0073] Figure 8 The binding kinetics of the anti-rabbit IgG multivalent linker measured using biolayer interferometry are shown, demonstrating little dissociation. The multivalent linker was adjusted stepwise from 2.5 nM to 0.3 nM on immobilized rabbit IgG.

[0074] Fig. 9Results of dynamic light scattering (DLS) analysis are shown, indicating uniform labeling of antibodies using multivalent linkers. Test samples included: rabbit IgG primary antibody alone, or in complex with anti-rabbit IgG multivalent linkers or two conventional secondary antibodies (in Fab2 format or whole IgG format). The results showed that the multivalent linker had no effect on the oligomeric state of the primary antibody.

[0075] Fig.10 is a schematic diagram showing an exemplary scheme for labeling antibodies using multivalent linkers.

[0076] Figure 11A-11C The results of co-staining HeLa cells using two mouse IgG1 primary antibodies with multivalent linkers are shown. Anti-mouse IgG1 multivalent linkers conjugated to 555 dye and anti-mouse IgG1 multivalent linkers conjugated to 647 dye were used to label mouse IgG1 primary antibodies against HSP60 (mitochondria, red) and anti-GORASP2 (Golgi, green). DAPI was used to stain the nucleus (blue). Fig.11A Micrographs of individual channels are shown. Fig. 11B Merged images of all three channels are shown. Fig. 11C The cross-section indicated in 11B (white straight line) was calculated using the ImageJ plugin RGB Analyzer. The confocal image was acquired using a 100x oil objective.

[0077] Fig.12 Labeling of primary antibodies over a wide range of concentrations is shown. Primary antibodies at the indicated concentrations were labeled using multivalent linkers and used for staining of HeLa cells.

[0078] Fig.13A Results showing the stability of the complex of mouse IgG1 primary antibody with a multivalent linker. Here, seven mouse IgG1 primary antibodies were labeled with a multivalent linker for mouse IgG1 and stored at 4°C (a quencher was also added during storage). After 7 days, 28 days, and 3 months, HeLa cells were stained with an appropriate amount of the complex, including a freshly labeled positive control. The IF signal at each time point was normalized relative to the IF signal of the freshly prepared sample, and the normalized IF signal was shown to be 70% to 100% over the longest test period of 3 months. Fig. 13B Immunofluorescence images demonstrating the stability of multivalent linker staining are shown. HeLa cells were stained with the indicated primary antibodies and multivalent linkers and imaged the same day (day 0) or 42 days later.

[0079] Fig.14A and Fig. 14B Multiplex immunostaining images using multivalent linkers are shown. Fig.14AImages of PFA-fixed HeLa cells stained with the following mouse IgG1 primary antibodies labeled with anti-mouse IgG1 multivalent linkers are shown: anti-Lamin B1 (green), anti-HSP60 (red), and anti-GORASP2 (cyan). Confocal images were acquired using a 100x oil objective and post-processed. Fig. 14B Shown are multiplex images of PFA-fixed HeLa cells stained with the following rabbit primary antibodies labeled with an anti-rabbit IgG multivalent linker: anti-TDP43 (green), anti-TOM20 (red), anti-Lamin B1 (magenta), and anti-CD147 (cyan).

[0080] Fig.15A and Fig. 15B Images of multiplex immunostaining using multivalent linkers against different isoforms or together with chemically conjugated primary antibodies are shown. Fig.15A Images of PFA-fixed HeLa cells stained with the following primary antibodies labeled with multivalent linkers are shown: rabbit polyclonal IgG anti-TOM70 (green), mouse IgG2a anti-GNL3 (red), and mouse IgG2a anti-β-actin (magenta). Cell nuclei are in cyan.

[0081] Fig. 15B Images of PFA-fixed HeLa cells stained with mouse IgG2 anti-GLN3 chemically conjugated to 594 dye and with the following mouse IgG2a primary antibodies labeled with anti-mouse IgG2a multivalent linkers: anti-tubulin (green) and anti-Lamin A / C (magenta) are shown. Confocal images were acquired with a 100x oil objective and post-processed.

[0082] Fig.16A and Fig. 16B Shown are images of multiplex immunostaining of tissues using multivalent linkers. Fig.16A Shown are images of FFPE human kidney sections stained with: rabbit polyclonal IgG anti-calbindin labeled with anti-rabbit IgG multivalent linker conjugated to 555 dye (yellow), mouse IgG1 anti-ACE2 labeled with anti-mouse IgG1 multivalent linker conjugated to 647 dye (magenta), 488-conjugated rabbit polyclonal anti-podocalyxin (green), and DAPI (blue). Fig. 16B Shown are images of FFPE rat brain tissue sections stained with: mouse IgG1 anti-NeuN labeled with anti-mouse IgG1 multivalent linker conjugated to 647 dye (magenta), mouse IgG1 anti-TUBB3 labeled with anti-mouse IgG1 multivalent linker conjugated to 555 dye (orange), and 488-conjugated mouse IgG2a anti-GFAP (green).

[0083] Fig.17Imaging results of CyCIF multiplex assays using multivalent linkers are shown. Fixed HeLa cells were immunostained for three cycles using the indicated primary antibodies labeled with 750-conjugated multivalent linkers and bleached using 4.5% H2O2 + 24 mM NaOH in PBS (room temperature for 1 hour under light).

[0084] Fig.18 Results of flow cytometry leakage assays of multivalent linkers are shown. PBMCs were stained with: mouse IgG1 isotype control labeled with anti-mouse IgG1 multivalent linker (left), mouse IgG1 anti-CD3 monoclonal antibody labeled with anti-mouse IgG1 multivalent linker (center), or isotype control labeled with anti-mouse IgG1 multivalent linker and treated with quencher in the presence of anti-CD3 antibody (right).

[0085] Fig.19 The results of flow cytometry multiplex analysis of surface markers of peripheral blood mononuclear cells (PBMCs) using primary antibodies of different isotypes are shown. Mouse IgG1 anti-CD45, mouse IgG2a anti-CD3, and mouse IgG2b a-C4 were stained with anti-mouse IgG1 multivalent linker conjugated to 488 dye, anti-IgG2a multivalent linker conjugated to 555 dye, or anti-IgG2b multivalent linker conjugated to 647 dye, respectively, and used to stain PBMCs.

[0086] Fig. 20 Results of multiplex flow cytometry analysis of surface markers of peripheral blood mononuclear cells (PBMCs) using primary antibodies of the same isotype are shown. Mouse IgG1 primary antibodies anti-CD3 (FITC), anti-CD4 (555 dye), anti-CD8 (647 dye), and anti-CD45 (750 dye) were labeled with multivalent linkers and used to stain PBMCs.

[0087] Fig.21 The results of flow cytometric analysis of intracellular markers of HEK293T cells are shown. Rabbit polyclonal antibodies against mitochondrial outer membrane proteins TOMM40 and TOMM20 and inner membrane proteins COX2 and mitofilin were labeled with multivalent linkers and HEK293T cells were stained.

[0088] Fig. 22Flow cytometry results are shown, demonstrating the compatibility of the multivalent linker with additives. Anti-CD3 primary antibodies stored in different concentrations of glycerol or bovine serum albumin (BSA) or in cell culture medium containing 15% fetal bovine serum (FBS) were labeled with a multivalent linker and used to stain T cell subsets of peripheral blood mononuclear cells (PBMCs). The primary antibody clone in the glycerol and cell culture medium experiments was mouse IgG1, while mouse IgG2a was used in the BSA titration (co-stained with mouse IgG1 anti-CD45).

[0089] Fig.23 Flow cytometry results are shown, demonstrating the compatibility of the multivalent linker in flow cytometry in the presence of additives. A primary mouse IgG1 anti-CD4 antibody was labeled with an anti-mouse IgG1 multivalent linker conjugated to 647 dye and used to stain the CD4+1 subset of peripheral blood mononuclear cells (PBMCs) in the presence of increasing concentrations of bovine serum albumin (BSA), fetal bovine serum, and the chelating agent EDTA.

[0090] Figure 24A-Figure 24C Results of multivalent linker labeling of hybridoma supernatants are shown. Fig.24A Hybridoma supernatants with varying amounts of mouse IgG1 anti-CD3 were simulated by adding the indicated amounts of primary antibodies to 1x RPMI medium plus 15% FBS, labeled with an anti-mouse IgG1 multivalent linker conjugated to 647 dye, and used to stain PBMCs. Fig. 24B Supernatants of hybridoma cells expressing mouse IgG2a anti-CD3 were diluted as indicated in IMDM medium plus 15% FBS, labeled with anti-mouse IgG2a multivalent linker conjugated to 647 dye, and used to stain PBMCs. As controls, the indicated amounts of purified antibodies were treated similarly. Fig.24C Hybridoma cells expressing mouse IgG2a anti-CD3 were seeded in 96-well plates at the indicated numbers. After 72 hours, the supernatant was labeled with anti-mouse IgG1 multivalent linker conjugated to 647 dye and used to stain PBMCs. DETAILED DESCRIPTION definition

[0091] All references cited are incorporated herein by reference in their entirety. In this application, unless otherwise noted, the techniques used can be found in any of the following well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification" in Methods in Enzymology (MP Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and Ambion 1998 catalog (Ambion, Austin, Tex.).

[0092] As used herein, the singular form "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0093] Unless the context clearly indicates otherwise, all embodiments of any aspect of the disclosure can be used in combination.

[0094] Unless the context clearly requires otherwise, throughout the specification and claims, the words "include", "comprising", etc. should be interpreted in an inclusive sense, rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to". In addition, the words "herein", "above", "below", and words of similar meaning, when used in this application, shall refer to this application as a whole and not to any particular parts of this application.

[0095] The term "about" refers to a quantity, level, value, number, frequency, percentage, size, size, amount, weight or length that varies by up to 10% relative to a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight or length, inclusive of the endpoints. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical values, unless it is clear from the context that the boundaries cannot be extended beyond certain points (e.g., below 0% or above 100%).

[0096] The term "affinity" refers to the strength of the interaction between an antigen and an antigen binding site of a binding agent (e.g., an antibody). Affinity can be determined, for example, using the following equation K A =[Ab:Ag] / [Ab][Ag]; Among them, K A = affinity constant; [Ab] = molar concentration of unoccupied binding sites on the binder; [Ag] = molar concentration of unoccupied binding sites on the target antigen; and [Ab:Ag] = molar concentration of the binder-target antigen complex. K A Describes the amount of binder-antigen complex present when equilibrium is reached. The time required for this process depends on the diffusion rate and is similar for each antibody. However, a high affinity antibody will bind a greater amount of antigen in a shorter period of time than a low affinity antibody. The K of the resulting antibody is A can vary and range from about 10 5 mol -1 to about 10 12 mol -1 or higher. K A Can be affected by factors including pH, temperature and buffer composition.

[0097] Antibody affinity can be measured using any means commonly used in the art, including but not limited to the use of biosensors, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI). The resonance unit is proportional to the degree of binding of a soluble ligand (e.g., a protein antigen) to an immobilized binding agent (or a soluble binding agent to an immobilized ligand such as a protein antigen). By determining the amount of binding of different known concentrations of a binding agent (e.g., an antibody) and a ligand (e.g., a protein antigen) at equilibrium, the equilibrium constant (K) can be calculated. A , K D ) and the dissociation and association rates (k off , k on ). A Formula K can be used A =k on / k off calculate.

[0098] Unless otherwise stated, affinity measurements (K A or K D ) were obtained from the dissociation / association rates (k off and k on ) is concluded.

[0099] The term "avidity" refers to the cumulative strength of multiple affinities of individual non-covalent binding interactions (such as between a multivalent linker and its antigen). Avidity is related to the affinity of individual immunoglobulin molecules in the population for a particular epitope, and is also related to the valence of the multivalent linker and the antigen. Thus, when compared to a monovalent molecule, the affinity of a multivalent binding molecule may appear higher, and the dissociation rate may appear slower, but the affinity and dissociation rate of each individual binding domain of the multivalent molecule are the same as the monovalent affinity or dissociation rate, respectively.

[0100] The term “k off " refers to the dissociation rate or dissociation constant, or the specific reaction rate at which a binding agent dissociates from a binding agent / antigen complex, measured in 1 / second (s -1 ).

[0101] The term “k on " is the association rate constant or on, or the specific reaction rate of a direct reaction or a complex formation reaction, measured in M -1 s -1 .

[0102] Unless otherwise stated, “k off " or "k on " Values ​​were obtained using biolayer interferometry (BLI) under the following conditions: phosphate buffered saline pH 7, 0.1% (m / v) BSA, 0.02% (v / v) Tween20, and 0.02% NaN3; all samples were set up in microplates (200 μl / well) at room temperature, and all experiments were run at 30°C, a shaking speed of 1000 rpm, and a recording rate of 5 Hz. The target antigen unit of the multivalent linker is immobilized on the biosensor, preferably, streptavidin immobilized to the biosensor via a biotin tag when applicable.

[0103] "Equilibrium dissociation constant" or "dissociation constant" or "K D "You can use the formula K D =k off / k on Calculate. K D is the k between the binder and its antigen off / k on The ratio of K D and affinity (K A) is negatively correlated. D The lower the value (lower peptide concentration), the higher the affinity of the antibody. The K of most antibody-derived antigen-binding domains is D Values ​​in the low micromolar range (10 -6 M) to nanomolar (10 -7 Up to 10 -9 High affinity antigen binding domains are generally considered to have low nanomolar range (10 -9 K in M) D , and the extremely high affinity K D In picomolar (10 -12 M) range is even lower (e.g. 10 -13 Up to 10 -14 In one embodiment, the apparent K of the peptides disclosed herein is D The range is about 10 -6 to about 10 -15 M, about 10 -7 to about 10 -15 M, about 10 -8 to about 10 -15 M, about 10 -9 to about 10 -15 M, about 10 -10 to about 10 -15 M, about 10 -11 to about 10 -15 M, about 10 -12 to about 10 -15 M, about 10 -13 to about 10 - 14 M, about 10 -13 to about 10 -15 M, or about 10 -14 to about 10 -15 M.

[0104] The multivalent linkers produced by the methods disclosed herein have high avidity, indicating that they bind tightly to the antigen. In some embodiments, the multivalent linkers of the present disclosure have very low apparent off-rates (koff) due to avidity effects.

[0105] The term "valent" refers to the presence of a specified number of binding sites of a binding agent (e.g., a multivalent linker). Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites in a binding agent, respectively (e.g., a multivalent linker of the present disclosure). A multivalent linker according to the present disclosure may be at least "bivalent," "trivalent," "tetravalent," or "hexavalent."

[0106] In some embodiments, the multivalent linker is divalent, trivalent, tetravalent, or hexavalent. In some embodiments, the multivalent linker is divalent. In some embodiments, the multivalent linker is trivalent. In some embodiments, the multivalent linker is tetravalent. In some embodiments, the multivalent linker is hexavalent.

[0107] The term "immunoglobulin" refers to a glycoprotein that may include at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen binding portion thereof. Each heavy chain has a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region may comprise three domains, C H 1. C H 2 and C H 3. Each light chain has a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region contains a domain C L . V H and V L The V region can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). CDRs contain most of the residues responsible for the specific interaction of an antibody with an antigen. Each V H and V L There are three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with epitopes of antigens. The term "immunoglobulin" may also include two heavy chains and no light chain, such as antibodies lacking light chains.

[0108] Each light chain of an immunoglobulin comprises an N-terminal variable (V) domain (VL) and a constant I domain (CL). Each heavy chain comprises an N-terminal V domain (VH), three or four C domains (CH), and a hinge region.

[0109] Immunoglobulins can be tetrameric glycosylated proteins composed of two light (L) chains of about 25 kDa each and two heavy (H) chains of about 50 kDa each. Two types of light chains can be found in immunoglobulins, called λ and κ. According to the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into five major classes: A, D, E, G, M and Y, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgM immunoglobulins are composed of 5 basic heterotetrameric units together with an additional polypeptide called J chain, and contain 10 antigen binding sites, while IgA immunoglobulins contain 2-5 basic 4-chain units, which can be polymerized to combine with J chains to form a multivalent assembly (assemblage).

[0110] An immunoglobulin "specifically for" or "specifically binds" (used interchangeably herein) a target (e.g., HA) is a term familiar to the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than it does with an alternative cell or substance. An immunoglobulin "specifically binds" to a particular protein or substance if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to an alternative specific protein or substance. For example, an immunoglobulin that specifically or preferentially binds to HA is an immunoglobulin that binds to HA with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other proteins. An immunoglobulin that specifically binds to a first protein or substance may or may not specifically or preferentially bind to a protein, cell, or substance. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding. Typically, but not necessarily, references to binding mean specific binding.

[0111] In some embodiments, "target antigen" refers to an antibody domain to which a multivalent linker of the present disclosure binds.

[0112] As used herein, the term "target antigen unit" refers to a single unit (e.g., a molecule, peptide, or other structurally distinguishable particle) comprising one or more epitopes bound by a multivalent linker of the present disclosure. In some embodiments, the binding agent (e.g., a primary antibody) is a target antigen unit.

[0113] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are identical over the entire residue (e.g., nucleotide or amino acid) alignment window. The "identity score" of the alignment segment of a test sequence and a reference sequence is the number of identical residues that the two aligned sequences have divided by the total number of residues in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The "identity percentage" is the identity score multiplied by 100. Sequence comparisons to determine percent identity can be accomplished by many well-known methods, including, for example, by using mathematical algorithms, such as those in the BLAST suite of sequence analysis programs.

[0114] In some embodiments, the identity of related polypeptide or nucleic acid sequences can be readily calculated by any method known to those of ordinary skill in the art. The "percent identity" of two sequences (e.g., nucleic acid or amino acid sequences) can be determined, for example, using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into Altschul et al., J. Mol. Biol. 215:403-10, 1990. and Program (version 2.0). Protein searches can be performed, for example, using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the proteins described herein. When there is a gap between the two sequences, a Gapped sequence search can be used, for example, as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402, 1997. In use and Gapped When you use a program, you can use the corresponding program (for example, and ) or the parameters may be appropriately adjusted according to the understanding of a person of ordinary skill in the art.

[0115] Another local alignment technique that can be used is based on, for example, the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). A general global alignment technique that can be used is, for example, the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453), which is based on dynamic programming.

[0116] Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed, which is said to produce global alignments of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. In some embodiments, the identity of two polypeptides is determined by aligning the two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one of the amino acid sequences. In some embodiments, the identity of two nucleic acids is determined by aligning the two nucleotide sequences and calculating the number of identical nucleotides, and dividing by the length of one of the nucleic acids.

[0117] For multiple sequence alignment, Clustal (Sievers et al., Mol Syst Biol. 2011 Oct 11; 7:539). Unless otherwise indicated, the term "sequence identity" in the claims refers to the sequence identity determined by Clustal Sequence identities were calculated using default parameters.

[0118] The term "therapeutic agent" refers to a moiety conjugated directly or indirectly to a multivalent linker that has a specific biological effect. For example, a therapeutic agent may include, but is not limited to, a chemotherapeutic agent.

[0119] The term "reporter molecule" refers to a detectable moiety that is directly or indirectly conjugated to a multivalent linker. Reporter molecules include, but are not limited to, fluorescent reporters, chemiluminescent reporters, radioactive reporters, and magnetic reporters. Limitations of Traditional Immunoassays

[0120] Detection of a protein of interest by antibody usually requires a primary antibody and a secondary antibody conjugated to a reporter molecule. For example, the primary antibody is usually obtained from a specific animal species, such as a rabbit, and is used to bind the protein of interest. The secondary antibody specifically recognizes the primary antibody and usually carries an attached label that is then used to detect the primary antibody (ideally bound to the epitope of interest). The secondary antibody is usually derived from another species than the primary antibody.

[0121] Labeling with reporter molecules on secondary antibodies is usually performed using non-directional chemistry. Therefore, the number of reporter molecules present in each single secondary antibody is not precisely known. There is an average of between 0.5 and about 3.5 reporter molecules on each secondary antibody, and this value may vary from one distributor to another and from one batch to another. Therefore, this variability in immunoassays leads to reduced reproducibility. For example, an average of 0.5 to about 3.5 reporter molecules on each secondary antibody means that some secondary antibodies have no reporter molecules (no detection), while some have up to 5 or more reporter molecules (increased detection).

[0122] Another traditional technique is to directly label the primary antibody with a reporter molecule, which is also usually performed using non-directed chemistry (i.e., random). Reporter molecules are usually not coupled directly to the primary antibody due to the risk of inactivating the binding ability of the antibody. This risk is particularly high on monoclonal antibodies because the addition of a reporter molecule to the paratope (epitope binding region on the antibody) will completely impair the antibody binding function of all molecules. Polyclonal antibodies may not be completely inactivated by coupling due to the possibility that some antibody molecules are not affected by the applied chemistry, but the affinity of the labeled antibody may vary, thereby introducing further variability in the assay. In addition, chemical labeling of primary antibodies, such as by NHS ester chemistry, is hindered by incompatible buffer substances and additives commonly used in antibody formulations (such as TRIS, BSA or azide), or can result in loss of activity.

[0123] One disadvantage of classical immunoassays is that the complexes of standard primary and secondary antibodies tend to form clusters. In addition, the reporter molecule is displaced between 15-25 nm relative to the desired target molecule. Since the resolution of existing microscopes is about 5 nm, the use of primary and secondary antibodies can result in errors of 10-20 nm in the true position of the target being studied in all three dimensions.

[0124] Another disadvantage of classical immunoassays is the inability to use multiple primary antibodies from the same species in the same experiment (i.e., multiplexing is not possible). Specifically, the cross-reactivity and high k of traditional secondary antibodies offRates make it difficult / impossible to multiplex primary antibodies from the same species, as the labeled secondary antibody may dissociate from its original target and bind to another primary antibody (i.e., signal leakage). This inability to multiplex increases the time and cost of experiments and often forces researchers to use inferior secondary antibodies from different species to avoid the aforementioned leakage problem.

[0125] Another disadvantage of classical immunoassays is the typical production method of antibodies. For example, the production of primary and secondary antibodies requires in vivo immunization in individual animals, followed by serum collection. There are great differences in the immune response between individual animals, resulting in variability between batches. It is expensive to maintain immune animals and can cause animal welfare issues (Reardon, S. US government issues historic $3.5-million fine over animal welfare. Nature (2016)). Hybridomas can be prepared for specific antibodies to reduce variability; however, these methods are time-consuming and costly for most antibodies.

[0126] Classical immunoassays also involve time-consuming and error-prone experimental protocols, which are further limitations of classical immunoassay methods. Current protocols typically apply the primary antibody for a specific time, usually ranging from 0.5 to 24 hours, and then wash away the excess primary antibody that did not find any target. After washing, the secondary antibody is incubated for a specific time, usually ranging from 0.5 to 18 hours, and then washed several times, usually for 10 to 30 minutes, to ensure that no free secondary antibody labeled with the reporter molecule remains in the preparation. The additional washing steps associated with traditional immunoassays are prone to errors, which can easily lead to a decrease in the primary signal, thereby hindering the experimental interpretation. Multivalent Linkers of the Disclosure

[0127] In some embodiments, the present disclosure solves the above-mentioned problems associated with traditional immunoassays and processing. Specifically, in some embodiments, the present disclosure teaches a multivalent linker that specifically binds to a target antigen, the multivalent linker comprising: a) a plurality of peptide binding arms, each binding arm capable of binding to an epitope in the same target antigen; and b) a linker segment operably covalently linked to the plurality of peptide binding arms. In some embodiments, the linker segment is long enough to maintain the peptide binding arm at a distance factor of c) preselected, which is designed to reduce or avoid cross-linking with other target antigen units. In some embodiments, the multivalent linker of the present disclosure further comprises d) a reporter or effector.

[0128] In some embodiments, advantages of the multivalent linkers of the present disclosure include: (1) high specificity for their targets, (2) the ability to be labeled with fluorophores or other selected labels, and (3) very low dissociation rates (k off ) binds to the target. For example, 10 -5 s -1 K off This results in only 5% dissociation within 90 min (approximately the time frame of a typical staining experiment). Figure 7 ). In fact, such low-k off This will allow labeling of the primary antibody while minimizing the risk of cross-staining due to dissociation and rebinding of the multivalent linker to a different primary antibody of the same species or isotype.

[0129] As discussed above, one problem with immunoassays is that the same species of primary antibodies cannot be used in the same assay. The present disclosure provides a solution to this problem through a labeling step that binds the multivalent linker to the primary antibody in a safe manner that preserves the paratope of the primary antibody. That is, in some embodiments, the multivalent linker of the present disclosure has excellent apparent k off The rate is increased, allowing the multivalent linker to bind the primary antibody without the harsh conditions typically required in direct labeling procedures.

[0130] The multivalent linker labeling step further allows the use of multiple primary antibodies from the same species in the same assay. In many cases, the "best" antibody in a particular field of research will be known and used. Therefore, classical immunoassays are limited to using antibodies from different species to label different target proteins. This results in experiments not being able to be performed due to a lack of a suitable combination of antibodies from different species or because the process of testing antibodies can cost thousands of dollars and take weeks to find the right antibody.

[0131] Another advantage of the present disclosure over the prior art is the size of the multivalent linker. For example, a typical secondary antibody is about 150 kDa. The larger size of the secondary antibody introduces the above problems, i.e., each secondary antibody is evenly distributed with 0.5 to about 3.5 reporter molecules and is displaced about 15-25 nm relative to the target molecule. In some embodiments, the multivalent linker of the present disclosure is much smaller than a typical antibody. For example, the divalent peptide of the present disclosure can be about 25-35 kDa, thereby reducing these problems.

[0132] Another advantage of the multivalent linker of the present disclosure relates to how to identify and produce the multivalent linker. Traditional secondary antibodies require in vivo immunization in individual animals, followed by serum collection. Therefore, in addition to causing animal welfare issues, traditional techniques also introduce batch variability. The in vitro techniques disclosed herein avoid the need for in vivo production of these consumables, reducing batch variability and the need to raise immune animals. Therefore, in some embodiments, the multivalent linker of the present disclosure exhibits characteristics that are superior to antibodies produced by traditional means.

[0133] Traditional immunoassays have multiple washing steps, which can increase the leakage of primary or secondary antibodies. For example, the application range of a primary antibody is 0.5 to 24h, followed by several washing steps, then the secondary antibody is applied, ranging from 0.5 to 18h, and finally another washing step is performed. The other washing steps associated with traditional immunoassays can cause the primary signal to weaken, thereby hindering experimental interpretation. The multivalent linker of the present disclosure can bypass several washing steps in these washing steps in the following manner: by very strong binding (e.g., non-covalent binding) to label a primary antibody. In addition, the present disclosure can reduce the amount of time required for the assay, because the secondary antibody step can be omitted and the connection step taught herein is adopted instead.

[0134] The multivalent linkers of the present disclosure allow rapid labeling of primary antibodies for use in multiple immunostaining techniques such as IF, IHC, flow cytometry, and Western blotting. Importantly, labeling with a multivalent linker is independent of the formulation or purification grade of the primary antibody. In addition, it is a highly scalable method because antibody amounts ranging from sub-microgram amounts to any amount can be labeled. This scalability may bring significant cost benefits to laboratories.

[0135] Additional information regarding various aspects of the multivalent linkers disclosed herein is provided herein. Joint section

[0136] In some embodiments, the disclosure provides a multivalent linker comprising two or more peptide binding arms covalently linked to a linker segment. In some embodiments, the disclosure provides a multivalent linker comprising a first peptide binding arm; a second peptide binding arm; and a linker segment disposed between the first peptide binding arm and the second peptide binding arm.

[0137] In some embodiments, the joint segment of the present disclosure is a polymer molecule comprising a repeating monomer structure. Thus, in some embodiments, the multivalent joint comprises the formula (peptide binding arm)-(joint segment)-(peptide binding arm). In some embodiments, the joint segment has two reactive ends to bind to separate peptide binding arms. In other embodiments, the joint segment has three or more reactive ends, each of which is capable of binding to its own peptide binding arm. In some embodiments, there is more than one joint segment, each of which is located between two peptide binding arms.

[0138] In some embodiments, the joint segment described herein is designed to connect (e.g., engage, link) two peptide binding arms, wherein the joint segment is not usually located between the two binding arms in nature. In the context of the present disclosure, the phrase "connection" or "engagement" or "connection" generally refers to a functional connection between two adjacent or neighboring amino acid sequences to produce a multivalent joint that does not exist in nature. In certain embodiments, connection can be used to refer to, for example, the covalent connection of the amino acid sequence of one or more peptide binding arms. Typically, the peptide binding arms connected are continuous or adjacent to each other and maintain their respective operability and function when engaged. In some embodiments, the peptide binding arms included in the multivalent joint disclosed herein are connected by interspersed joint segments. Such joint segments can provide the required flexibility to allow the peptide binding arms in the multivalent joint to achieve the desired expression, activity and / or conformational positioning.

[0139] In some embodiments, the linker segment comprises amino acids and / or consists essentially of amino acids. Typical amino acid linker segments between two protein moieties are generally designed to be flexible, or to intersperse a structure, such as an alpha helix. In some embodiments, the terms "linker," "linker segment," and "spacer" are used interchangeably.

[0140] In some embodiments, the linker segment may employ any one or more naturally occurring amino acids, one or more non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimetics described elsewhere herein and known in the art. Certain amino acid sequences that can be used as linker segments include Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA.83:8258-8262, 1986; those disclosed in U.S. Pat. Nos. 4,935,233 and 4,751,180. In some embodiments, the present disclosure teaches peptide linker segment sequences containing Gly, Ser, and / or Asn residues. In some embodiments, neutral amino acids (such as Thr and Ala) may also be used in peptide linker segment sequences if desired.

[0141] The linker segment peptide sequence can be any suitable length to connect one or more target proteins, and is preferably designed to be flexible enough to allow the correct folding and / or function and / or activity of one or both peptides it connects. In some embodiments, the length of the linker segment peptide along its longest axis is no more than 3, no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95 or no more than 100 amino acids, including all ranges and subranges therebetween. In some embodiments, the length of the linker segment peptide along its longest edge can be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, or 100 amino acids, including all ranges and subranges therebetween.

[0142] In some embodiments, the linker segment comprises at least 10 and no more than 60 amino acids, at least 10 and no more than 55 amino acids, at least 10 and no more than 50 amino acids, at least 10 and no more than 45 amino acids, at least 10 and no more than 40 amino acids, at least 10 and no more than 35 amino acids, at least 10 and no more than 30 amino acids, at least 10 and no more than 25 amino acids, at least 10 and no more than 20 amino acids, or at least 10 and no more than 15 amino acids. In some embodiments, the linker segment comprises at least 15 and no more than 60 amino acids, at least 15 and no more than 55 amino acids, at least 15 and no more than 50 amino acids, at least 15 and no more than 45 amino acids, at least 15 and no more than 40 amino acids, at least 15 and no more than 35 amino acids, at least 15 and no more than 30 amino acids, at least 15 and no more than 25 amino acids, or at least 15 and no more than 20 amino acids. In some embodiments, the linker segment comprises at least 20 and no more than 60 amino acids, at least 20 and no more than 55 amino acids, at least 20 and no more than 50 amino acids, at least 20 and no more than 45 amino acids, at least 20 and no more than 40 amino acids, at least 20 and no more than 35 amino acids, at least 20 and no more than 30 amino acids, or at least 20 and no more than 25 amino acids. In some embodiments, the linker segment comprises at least 25 and no more than 60 amino acids, at least 25 and no more than 55 amino acids, at least 25 and no more than 50 amino acids, at least 25 and no more than 45 amino acids, at least 25 and no more than 40 amino acids, at least 25 and no more than 35 amino acids, or at least 25 and no more than 30 amino acids. In some embodiments, the linker segment comprises at least 30 and no more than 60 amino acids, at least 30 and no more than 55 amino acids, at least 30 and no more than 50 amino acids, at least 30 and no more than 45 amino acids, at least 30 and no more than 40 amino acids, or at least 30 and no more than 35 amino acids. In some embodiments, these are the number of amino acids along the longest axis of the linker segment peptide sequence.

[0143] In some embodiments, in a polypeptide composition comprising a linker segment, the 5′ end (e.g., terminus) of the linker segment peptide sequence (e.g., amino acid sequence) is adjacent to and covalently linked to the 3′ end of one peptide sequence (e.g., full-length protein or protein domain, fragment or variant), and, further, the 3′ end of the linker segment amino acid sequence is adjacent to and covalently linked to the 5′ end of another peptide sequence.

[0144] In some embodiments, the linker segment comprises glycine. In some embodiments, the linker segment comprises glycine, alanine, serine and / or threonine amino acid residues. In some embodiments, the linker segment comprises glycine, alanine and / or serine amino acid residues. In some embodiments, the linker segment comprises glycine, threonine and / or serine amino acid residues. In some embodiments, the linker segment comprises glycine, alanine and / or threonine amino acid residues. In some embodiments, the linker segment comprises glycine and / or alanine amino acid residues. In some embodiments, the linker segment comprises glycine and / or serine amino acid residues. In some embodiments, the linker segment comprises glycine and / or threonine amino acid residues.

[0145] In some embodiments, the linker segment comprises glycine. In some embodiments, at least 20% of the amino acids in the linker segment are glycine. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the amino acids in the linker segment are glycine. In some embodiments, at least 50% of the amino acids in the linker segment are glycine. In some embodiments, at least 60% of the amino acids in the linker segment are glycine. In some embodiments, at least 70% of the amino acids in the linker segment are glycine. In some embodiments, at least 80% of the amino acids in the linker segment are glycine. In some embodiments, at least 90% of the amino acids in the linker segment are glycine. In some embodiments, 20%-90% of the amino acids in the linker segment are glycine. In some embodiments, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of the amino acids in the linker segment are glycine. In some embodiments, 20%-40%, 30%-50%, 40%-60%, 50%-70%, 60%-80%, or 70%-90% of the amino acids in the linker segment are glycine. In some embodiments, 20%-50%, 30%-60%, 40%-70%, 50%-80%, or 60%-90% of the amino acids in the linker segment are glycine. In some embodiments, 60%-90% of the amino acids in the linker segment are glycine.

[0146] In some embodiments, the joint segment comprises alanine. In some embodiments, at least 5% of the amino acids in the joint segment are alanine. In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the amino acids in the joint segment are alanine. In some embodiments, 5%-15%, 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, or 40%-50% of the amino acids in the joint segment are alanine. In some embodiments, 5%-25%, 10%-30%, 15%-35%, 20%-40%, 25%-45%, or 30%-50% of the amino acids in the joint segment are alanine. In some embodiments, 10%-30%, 15%-25%, or about 20% of the amino acids in the linker segment are alanine.

[0147] In some embodiments, the linker segment comprises (i) glycine and (ii) alanine. In some embodiments, the ratio of glycine to alanine is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about 10:1. In some embodiments, the ratio of glycine to alanine is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 8.5:1, at least 9:1, at least 9.5:1, or at least 10:1. In some embodiments, the ratio of glycine to alanine is at most 1:1, at most 1.5:1, at most 2:1, at most 2.5:1, at most 3:1, at most 3.5:1, at most 4:1, at most 4.5:1, at most 5:1, at most 5.5:1, at most 6:1, at most 6.5:1, at most 7:1, at most 7.5:1, at most 8:1, at most 8.5:1, at most 9:1, at most 9.5:1, or at most 10:1.

[0148] In some embodiments, the joint segment comprises serine and / or threonine. In some embodiments, at least 5% of the amino acids in the joint segment are serine and / or threonine. In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of the amino acids in the joint segment are serine and / or threonine. In some embodiments, 5%-15%, 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45% or 40%-50% of the amino acids in the joint segment are serine and / or threonine. In some embodiments, 5%-25%, 10%-30%, 15%-35%, 20%-40%, 25%-45%, or 30%-50% of the amino acids in the linker segment are serine and / or threonine. In some embodiments, 10%-30%, 15%-25%, or about 20% of the amino acids in the linker segment are serine and / or threonine. In some embodiments, this amino acid is serine. In some embodiments, the ratio of serine to threonine is at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:1.

[0149] In some embodiments, the linker segment comprises (i) glycine and (ii) serine and / or threonine. In some embodiments, the ratio of glycine to serine and / or threonine is about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about 10:1. In some embodiments, the ratio of glycine to serine and / or threonine is at least 1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 8.5:1, at least 9:1, at least 9.5:1, or at least 10:1. In some embodiments, the ratio of glycine to serine and / or threonine is at most 1:1, at most 1.5:1, at most 2:1, at most 2.5:1, at most 3:1, at most 3.5:1, at most 4:1, at most 4.5:1, at most 5:1, at most 5.5:1, at most 6:1, at most 6.5:1, at most 7:1, at most 7.5:1, at most 8:1, at most 8.5:1, at most 9:1, at most 9.5:1, or at most 10:1.

[0150] In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K), and arginine (R). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K), and arginine (R). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), alanine (A), glutamate (E), aspartic acid (D), lysine (K), and arginine (R). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), glutamate (E), aspartic acid (D), lysine (K), and arginine (R). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), serine (S), threonine (T), glutamate (E), aspartic acid (D), and lysine (K). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), alanine (A), glutamate (E), aspartic acid (D), and lysine (K). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), glutamate (E), aspartic acid (D), and lysine (K). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), or threonine (T). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acids in the linker segment are glycine (G), serine (S), or threonine (T).In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the joint segment are glycine (G) or serine (S). In some embodiments, such amino acids account for at least 70% of the amino acids in the joint segment. In some embodiments, such amino acids account for at least 75% of the amino acids in the joint segment. In some embodiments, such amino acids account for at least 80% of the amino acids in the joint segment. In some embodiments, such amino acids account for at least 85% of the amino acids in the joint segment. In some embodiments, such amino acids account for at least 90% of the amino acids in the joint segment. In some embodiments, such amino acids account for at least 95% of the amino acids in the joint segment. In some embodiments, such amino acids account for 100% of the amino acids in the joint segment.

[0151] In some embodiments, the amino acids can be alternated / repeated in any manner consistent with the linker segment remaining functional (e.g., resulting in one or more expressed and / or active polypeptides). In some embodiments, the linker segment can be alternated / repeated in any manner, including the formula (G)nX (SEQ ID NO: 532), wherein n is 1-100, and X is any amino acid, such as alanine, serine, or glycine. For example, the amino acids in the linker segment can be repeated every other 1 (e.g., GAGA (SEQ ID NO:510), GSGS (SEQ ID NO:511)), every other 2 (e.g., GGAGGA (SEQ ID NO:512), GGSGGS (SEQ ID NO:513)), every other 3 (e.g., GGGAGGGA (SEQ ID NO:514), GGGSGGGS (SEQ ID NO:515)), every other 4 (e.g., GGGGAGGGGA (SEQ ID NO:516), GGGGSGGGGS (SEQ ID NO:517)), every 5, every 6, every 7, every 8, every 9, or every 10 or more amino acids, or the amino acids can be repeated in any combination of the above items.

[0152] In some embodiments, the amino acid is repeated every four amino acids, and the linker segment consists of one or more glycine repeats. In some embodiments, the linker segment comprises the formula (G4X)n, where n represents the number of repeats, and X is any amino acid, such as alanine, serine, or glycine. For example, the linker segment can consist of GGGGA (G4A) (SEQ ID NO: 518) or GGGGS (G4S) repeats (SEQ ID NO: 519).

[0153] In some embodiments, the linker segment sequence is GGGGAGGGGA(G4A)2 (SEQ ID NO:520), GGGGAGGGAGGGGA(G4A)3 (SEQ ID NO:521), GGGGAGGGGAGGGGAGGGGA(G4A)4 (SEQ ID NO:522), GGGGAGGGGAGGGGAGGGGAGGGGA(G4A)5 (SEQ ID NO: 523), GGGGAGGGGAGGGGAGGGGAGGGGAGGGGA (G4A) 6 (SEQ ID NO: 524) or GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGA (G4A) 7 (SEQ ID NO: 525).

[0154] In some embodiments, the linker segment sequence is GGGGSGGGGS(G4S)2 (SEQ ID NO:526), ​​GGGGSGGGGSGGGGS(G4S)3 (SEQ ID NO:527), GGGGSGGGGSGGGGSGGGGS(G4S)4 (SEQ ID NO:528), GGGGSGGGGSGGGGSGGGGSGGGGS(G4S)5 (SEQ ID NO:528) NO:529), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(G4S)6 (SEQ ID NO:530) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(G4S)7 (SEQ ID NO:531).

[0155] In some embodiments, the linker segment sequence comprises or consists of an amino acid sequence of GSTSGSGKSSEGKGEGSTSGSGKSG (SEQ ID NO: 495), or an amino acid sequence having at most 1, at most 2, at most 3, at most 4, or at most 5 amino acid mutations (additions, deletions, or substitutions) relative thereto. Distance Factor

[0156] The present invention is based in part on the unexpected discovery of the inventors that the peptide binding arms of the multivalent linkers of the present disclosure can all bind to the same target without cross-linking with other molecules. This results in excellent binding properties due to the enhanced avidity achieved by the apparent (i.e., synergistic) affinity of the multiple peptide binding arms to the target. In this binding mode, it is believed that one binding arm of the bivalent peptide binds to a first epitope of multiple epitopes in the target, followed by an additional (e.g., second) binding arm engaging an epitope of the same target. To do this, the linker segments between the binding arms need to be long enough and flexible to reach additional epitopes in the same target.

[0157] Without wishing to be bound by any one theory, it is believed that if the linker segment between the binding arms is too short, the additional binding arms may not be able to reach additional epitopes on the target (e.g., epitopes within the same primary antibody). In this case, the additional binding arms may eventually engage with different targets (e.g., another primary antibody), resulting in cross-linking, thereby causing the targets to aggregate. Therefore, in some embodiments, the disclosure provides a minimum distance factor between binding arms.

[0158] In some embodiments, the distance is measured from the first amino acid of each peptide binding arm that is connected to the linker segment.

[0159] In some embodiments, the distance between the two peptide binding arms of the multivalent linker created by the linker segment is about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about Approx. 244 about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about about or about Any ranges and sub-ranges therebetween are included.

[0160] In some embodiments, the distance between the two peptide binding arms of the multivalent linker created by the linker segment is to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between to between or to between.

[0161] In some embodiments, the minimum distance factor between the two peptides of the multivalent linker reduces cross-linking. In some embodiments, cross-linking is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%, including all ranges and subranges therebetween.

[0162] In some embodiments, cross-linking is reduced by between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 10% and 15%, between 15% and 20%, between 20% and 25%, between 25% and 30%, between 30% and 35%, between 35% and 40%, between 40% and 45%, between 45% and 50%, between 50% and 55%, between 55% and 60%, between 60% and 65%, between 65% and 70%, between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%, including all ranges and subranges therebetween.

[0163] In some embodiments, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, or at most 50% of the multivalent linkers are cross-linked to different antibodies.

[0164] In some embodiments, between 0% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 10% and 15%, between 15% and 20%, between 20% and 25%, between 25% and 30%, between 30% and 35%, between 35% and 40%, between 40% and 45%, or between 45% and 50% of the multivalent linkers are cross-linked to different antibodies, including all ranges and subranges therebetween.

[0165] In some embodiments, when the distance between two peptides of a multivalent linker created by a linker segment is measured in angstroms, the distance is determined spatially, such as by crystallography (eg, X-ray crystallography) or 3-D modeling software.

[0166] In some embodiments, the distance between the two peptide binding arms of the multivalent linker generated by the linker segment is at most 1 (G4X) unit, at most 2 (G4X) units, at most 3 (G4X) units, at most 4 (G4X) units, at most 5 (G4X) units, at most 6 (G4X) units, at most 7 (G4X) units, at most 8 (G4X) units, at most 9 (G4X) units or at most 10 (G4X) units, including any ranges and subranges therebetween.

[0167] In some embodiments, the distance between the two peptide binding arms of the multivalent linker produced by the linker segment is at most 1 (G4A) unit, at most 2 (G4A) units, at most 3 (G4A) units, at most 4 (G4A) units, at most 5 (G4A) units, at most 6 (G4A) units, at most 7 (G4A) units, at most 8 (G4A) units, at most 9 (G4A) units or at most 10 (G4A) units, including any ranges and sub-ranges therebetween.

[0168] In some embodiments, the distance between the two peptide binding arms of the multivalent linker generated by the linker segment is at most 1 (G4S) unit, at most 2 (G4S) units, at most 3 (G4S) units, at most 4 (G4S) units, at most 5 (G4S) units, at most 6 (G4S) units, at most 7 (G4S) units, at most 8 (G4S) units, at most 9 (G4S) units or at most 10 (G4S) units, including any ranges and sub-ranges therebetween. Moiety for conjugation to heterologous molecules

[0169] In some embodiments, the multivalent linker comprises at least one moiety for conjugation to a heterologous molecule. In some embodiments, the at least one moiety is cysteine. In some embodiments, the at least one moiety is lysine. In some embodiments, the at least one moiety is biotin. In some embodiments, the biotinylation of the multivalent linker is achieved by chemical methods. In some embodiments, the biotinylation of the multivalent linker is achieved by enzymatic methods. In some embodiments, the biotinylation of the multivalent linker is achieved in vitro. In some embodiments, the biotinylation of the multivalent linker is achieved in vivo.

[0170] Exemplary methods of biotinylation, as well as other bioconjugation techniques, can be found in Hermanson GT (2008) Bioconjugate techniques, 2nd ed. San Diego (CA): Academic Press, the contents of which are incorporated by reference in their entirety for all purposes.

[0171] In some embodiments, the at least one moiety is streptavidin.

[0172] In some embodiments, the at least one moiety is an unnatural amino acid.

[0173] In some embodiments, the at least one portion comprises a functional group for conjugation by click chemistry. In some embodiments, the functional group includes a dibenzocyclooctyne group (DBCO), an azide, a tetrazine and / or a trans-cyclooctene (TCO). A description of click chemistry can be found in, for example, Devaraj and Finn, Chem. Rev. 2021, 121, 12, 6697-6698; and Hein et al., Pharm Res. October 2008; 25 (10): 2216-2230, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0174] In some embodiments, the multivalent linker comprises a moiety for conjugation to a heterologous molecule.

[0175] In some embodiments, the multivalent linker comprises two or more moieties for conjugation to a heterologous molecule. In some embodiments, the multivalent linker comprises two, three, four, five, six, seven, eight, nine, ten or more than ten moieties for conjugation to a heterologous molecule. In some embodiments, the multivalent linker comprises two moieties for conjugation to a heterologous molecule. In some embodiments, the two or more moieties are the same. In some embodiments, the two or more moieties are different.

[0176] In some embodiments, the linker segment of the multivalent linker comprises at least one moiety for conjugation to an exogenous molecule. In some embodiments, the linker segment of the multivalent linker comprises all moieties for conjugation to a heterologous molecule.

[0177] In some embodiments, the linker segment of the multivalent linker does not include any moiety for conjugation to a heterologous molecule.

[0178] In some embodiments, the peptide binding arm of the multivalent linker comprises at least one moiety for conjugation to a heterologous molecule. In some embodiments, the peptide binding arm of the multivalent linker comprises all moieties for conjugation to a heterologous molecule.

[0179] In some embodiments, the peptide binding arm of the multivalent linker does not comprise any moiety for conjugation to a heterologous molecule.

[0180] In some embodiments, the heterologous molecule is a reporter, an oligonucleotide, a moiety functionalized for click chemistry, or an effector. Exemplary Multivalent Linker Structures

[0181] In some embodiments, the peptides of the present disclosure are multivalent, comprising multiple peptide binding arms.

[0182] In some embodiments, the multivalent peptide comprises the formula (SEQ ID NO. 1-418)-(linker segment)-(SEQ ID NO. 1-418).

[0183] In some embodiments, the multivalent linker comprises the formula (SEQ ID NO.1-418)-(G4S) n -(SEQ ID NO. 1-418).

[0184] In some embodiments, the multivalent linker comprises the same peptide sequence.In some embodiments, the multivalent linker comprises the formula in Table 1. Table 1. Multivalent linkers containing the same peptide sequence

[0185] In some embodiments, the multivalent linker comprises different peptide sequences. In some embodiments, the multivalent linker comprises the formula in Table 2. Table 2. Multivalent linkers comprising different peptide sequences.

[0186] The multivalent linker of the present disclosure is at least "divalent", and can be at least "trivalent", "tetravalent", or "hexavalent". In some embodiments, the multivalent linker is divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent or higher (e.g., containing 2, 3, 4, 5, 6, 7 or more peptide binding arms selected from SEQ ID NO: 1-418).

[0187] For example, in some embodiments, a trivalent peptide of the present disclosure comprises the formula (peptide binding arm)-(linker segment)-(peptide binding arm 2)-(linker segment)-(peptide binding arm)

[0188] For example, in some embodiments, the trivalent peptide of the present disclosure comprises the formula (SEQ ID NO.1-418)-(G4S) n -(SEQ ID NO.1-418)-(G4S) n -(SEQ ID NO. 1-418).

[0189] In some embodiments, the multivalent linker of the present disclosure is less than 90 kDa, less than 85 kDa, less than 80 kDa, less than 75 kDa, less than 70 kDa, less than 65 kDa, less than 60 kDa, less than 55 kDa, less than 50 kDa, less than 45 kDa, less than 40 kDa, less than 35 kDa, less than 30 kDa, less than 25 kDa, less than 20 kDa, or less than 15 kDa.

[0190] In some embodiments, the multivalent linker is between 15-20 kDa, 20-25 kDa, 25-30 kDa, 30-35 kDa, 35-40 kDa, 40-45 kDa, 45-50 kDa, 50-55 kDa, 55-60 kDa, 60-65 kDa, 65-70 kDa, 70-75 kDa, 75-80 kDa, 80-85 kDa, or 85-90 kDa. K D and k off rate

[0191] The invention and development of the presently claimed multivalent linkers was based, in part, on the discovery that the additional binding strength provided by multiple peptide binding arms resulted in dramatic technical improvements in a variety of immunoassays and processes.

[0192] As discussed above, one of the important technical limitations of traditional dual antibody detection technology is signal leakage, which occurs due to the dissociation of antibodies from their primary binding agent and possible rebinding to another reagent.

[0193] In some embodiments, the multivalent linkers of the present disclosure achieve low apparent K D (pM) value (bound to target antigen unit).

[0194] In some embodiments, the apparent K of the multivalent linker is D(pM) less than 5,000, less than 4,000, less than 3,000, less than 2,000, less than 1,000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 50, less than 10 or less than 1 pM, including all ranges and subranges therebetween.

[0195] In some embodiments, the apparent K of the multivalent linker is D (pM) between 1 and 10, between 10 and 50, between 50 and 100, between 100 and 200, between 200 and 300, between 300 and 400, between 400-500, between 500 and 600, between 600 and 700, between 700 and 800, between 800 and 900, between 900 and 1,000, between 1,000 and 2,000, between 2,000 and 3,000, between 3,000 and 4,000, or between 4,000 and 5,000 pM, including all ranges and subranges therebetween.

[0196] In some embodiments, the multivalent linkers of the present disclosure achieve strong apparent k off For example, commercially available secondary antibodies can exhibit a rate of greater than 10 -4 s -1 K off Other monovalent peptide arms are also limited, such as Figure 1 and Figure 3 Thus, in some embodiments, the multivalent linkers of the present disclosure exhibit superior properties even over similar monovalent peptides (such as those disclosed in EP 3596464, the contents of which are incorporated herein in their entirety).

[0197] In some embodiments, the multivalent linkers of the present disclosure exhibit superior (lower) k off rate.

[0198] In some embodiments, the k of a multivalent linker disclosed herein is off (s -1 ) rate range is about 10 -4 to about 10 -10 , about 10 -5 to about 10 -10 , about 10 -6 to about 10 -10 , about 10 -7 to about 10 -10 , about 10 -8 to about 10 -10 , about 10 -9 to about 10-10 , or about 10 -10 .

[0199] In some embodiments, the multivalent linkers of the present disclosure exhibit less than 5% dissociation after 100 minutes (the average time for performing an antibody-based assay). off (s -1 ) rate is less than 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 or 10 -11 .

[0200] One of the advantages of the multivalent linkers disclosed in this application is that they achieve such a low k off Rate, while in some embodiments still do not need to be covalently linked to the target antigen (e.g., to a primary antibody). Therefore, at least in some embodiments, the multivalent linker disclosed in the present application is different from the covalent linker described in US11,123,440 and WO 2022115791.

[0201] In one embodiment, a multivalent linker disclosed herein exhibits a k off (s -1 ) rate is at most 10 -4 , up to 10 -5 , up to 10 -6 , up to 10 -7 , up to 10 -8 , up to 10 -9 or up to 10 -10 .

[0202] In some embodiments, the multivalent linker of the present disclosure is designed to specifically bind to an immunoglobulin of a single species, while not exhibiting cross-reactivity to immunoglobulins from other species. For example, in some embodiments, the multivalent linker of the present disclosure specifically binds to an immunoglobulin of a species selected from: human, guinea pig, mouse, rat, chicken, rabbit, goat, donkey, pig, horse, and cattle (e.g., cow), while not exhibiting cross-reactivity to immunoglobulins of other species.

[0203] As noted throughout this document, the multivalent linker of the present disclosure comprises a plurality of binding arms, which are believed to confer several advantages to the multivalent linker over other known antibody labeling techniques. In some embodiments, the multivalent linker of the present disclosure is not (and does not contain) an antigen binding fragment (FAB) molecule. That is, the multivalent linker of the present disclosure is different from and superior to FAB-based techniques, such as those disclosed on the World Wide Web at thermofisher.com / us / en / home / references / molecular-probes-the-handbook / antibodies-avidins-lectins-and-related-products / 56olyv-technology-versat ile-reagents-for-immunolabeling.html.

[0204] In some embodiments, the multivalent linker specifically binds human immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0205] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0206] In some embodiments, the multivalent linker specifically binds goat immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, human immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0207] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0208] In some embodiments, the multivalent linker specifically binds mouse immunoglobulins and does not cross-react with guinea pig immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0209] In some embodiments, the multivalent linker specifically binds donkey immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0210] In some embodiments, the multivalent linker specifically binds sheep immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0211] In some embodiments, the multivalent linker specifically binds porcine immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, horse immunoglobulins, bovine immunoglobulins, sheep immunoglobulins, or rabbit immunoglobulins.

[0212] In some embodiments, the multivalent linker specifically binds horse immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, sheep immunoglobulins, porcine immunoglobulins, or bovine immunoglobulins.

[0213] In some embodiments, the multivalent linker specifically binds bovine immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, sheep immunoglobulins, porcine immunoglobulins, or horse immunoglobulins.

[0214] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and does not cross-react with guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, sheep immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0215] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, rabbit immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, sheep immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0216] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or guinea pig immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0217] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or mouse immunoglobulins, and does not cross-react with guinea pig immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0218] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or rat immunoglobulins, and does not cross-react with mouse immunoglobulins, guinea pig immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0219] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or chicken immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, guinea pig immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0220] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or human immunoglobulins and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, guinea pig immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0221] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or goat immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, guinea pig immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0222] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or donkey immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, guinea pig immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0223] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or porcine immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, guinea pig immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0224] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or horse immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, guinea pig immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0225] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or bovine immunoglobulins, and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, guinea pig immunoglobulins, or sheep immunoglobulins.

[0226] In some embodiments, the multivalent linker specifically binds rabbit immunoglobulins and / or sheep immunoglobulins and does not cross-react with mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, guinea pig immunoglobulins, or bovine immunoglobulins.

[0227] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or mouse immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0228] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or rat immunoglobulins, and does not cross-react with rabbit immunoglobulins, mouse immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0229] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or chicken immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0230] In some embodiments, the multivalent linker specifically binds to guinea pig immunoglobulins and / or human immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, mouse immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0231] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or goat immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, mouse immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0232] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or donkey immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, mouse immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0233] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or porcine immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, mouse immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0234] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or horse immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, mouse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0235] In some embodiments, the multivalent linker specifically binds to guinea pig immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, mouse immunoglobulins, or sheep immunoglobulins.

[0236] In some embodiments, the multivalent linker specifically binds guinea pig immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, mouse immunoglobulins, or bovine immunoglobulins.

[0237] In some embodiments, the multivalent linker specifically binds mouse immunoglobulins and / or rat immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0238] In some embodiments, the multivalent linker specifically binds mouse immunoglobulins and / or chicken immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0239] In some embodiments, the multivalent linker specifically binds to mouse immunoglobulins and / or human immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, chicken immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0240] In some embodiments, the multivalent peptide specifically binds to mouse immunoglobulins and / or goat immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0241] In some embodiments, the multivalent linker specifically binds to mouse immunoglobulins and / or donkey immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, chicken immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0242] In some embodiments, the multivalent linker specifically binds to mouse immunoglobulins and / or porcine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, chicken immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0243] In some embodiments, the multivalent linker specifically binds mouse immunoglobulins and / or horse immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, chicken immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0244] In some embodiments, the multivalent linker specifically binds to mouse immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, chicken immunoglobulins, or sheep immunoglobulins.

[0245] In some embodiments, the multivalent linker specifically binds to mouse immunoglobulins and / or sheep immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, rat immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, chicken immunoglobulins, or bovine immunoglobulins.

[0246] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or chicken immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0247] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or human immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, chicken immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0248] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or goat immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0249] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or donkey immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, chicken immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0250] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or porcine immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0251] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or horse immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, chicken immunoglobulins, chicken immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0252] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, chicken immunoglobulins, or sheep immunoglobulins.

[0253] In some embodiments, the multivalent linker specifically binds rat immunoglobulins and / or sheep immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, chicken immunoglobulins, or bovine immunoglobulins.

[0254] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or human immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0255] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or goat immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, human immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0256] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or donkey immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, human immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0257] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or porcine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, donkey immunoglobulins, human immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0258] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or horse immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, human immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0259] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, or human immunoglobulins or sheep immunoglobulins.

[0260] In some embodiments, the multivalent linker specifically binds chicken immunoglobulins and / or sheep immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, human immunoglobulins, or bovine immunoglobulins.

[0261] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or goat immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0262] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or donkey immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, goat immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0263] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or porcine immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, goat immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0264] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or horse immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, goat immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0265] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, goat immunoglobulins, or sheep immunoglobulins.

[0266] In some embodiments, the multivalent linker specifically binds human immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, horse immunoglobulins, goat immunoglobulins, or bovine immunoglobulins.

[0267] In some embodiments, the multivalent linker specifically binds goat immunoglobulins and / or donkey immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, porcine immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0268] In some embodiments, the multivalent linker specifically binds to goat immunoglobulins and / or porcine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, donkey immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0269] In some embodiments, the multivalent linker specifically binds goat immunoglobulins and / or horse immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, porcine immunoglobulins, donkey immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0270] In some embodiments, the multivalent linker specifically binds goat immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, porcine immunoglobulins, horse immunoglobulins, donkey immunoglobulins, or sheep immunoglobulins.

[0271] In some embodiments, the multivalent linker specifically binds goat immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, porcine immunoglobulins, horse immunoglobulins, donkey immunoglobulins, or bovine immunoglobulins.

[0272] In some embodiments, the multivalent linker specifically binds to donkey immunoglobulins and / or porcine immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, horse immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0273] In some embodiments, the multivalent linker specifically binds donkey immunoglobulins and / or horse immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, porcine immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0274] In some embodiments, the multivalent linker specifically binds to donkey immunoglobulins and / or bovine immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, horse immunoglobulins, porcine immunoglobulins, or sheep immunoglobulins.

[0275] In some embodiments, the multivalent linker specifically binds donkey immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, horse immunoglobulins, or porcine immunoglobulins or bovine immunoglobulins.

[0276] In some embodiments, the multivalent linker specifically binds porcine immunoglobulins and / or horse immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, bovine immunoglobulins, or sheep immunoglobulins.

[0277] In some embodiments, the multivalent linker specifically binds porcine immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, horse immunoglobulins, or sheep immunoglobulins.

[0278] In some embodiments, the multivalent linker specifically binds porcine immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, horse immunoglobulins, or bovine immunoglobulins.

[0279] In some embodiments, the multivalent linker specifically binds horse immunoglobulins and / or bovine immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, porcine immunoglobulins, or sheep immunoglobulins.

[0280] In some embodiments, the multivalent linker specifically binds horse immunoglobulins and / or sheep immunoglobulins and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, or porcine immunoglobulins or bovine immunoglobulins.

[0281] In some embodiments, the multivalent linker specifically binds bovine immunoglobulins and / or sheep immunoglobulins, and does not cross-react with rabbit immunoglobulins, guinea pig immunoglobulins, mouse immunoglobulins, rat immunoglobulins, chicken immunoglobulins, human immunoglobulins, goat immunoglobulins, donkey immunoglobulins, or porcine immunoglobulins or horse immunoglobulins.

[0282] In some embodiments, the multivalent linker specifically binds to the constant region of an immunoglobulin heavy chain.

[0283] In some embodiments, the multivalent linker specifically binds to the hinge region of an immunoglobulin heavy chain.

[0284] In some embodiments, the multivalent linker specifically binds to the constant region of an immunoglobulin light chain. Peptide Binding Arms of the Disclosure

[0285] In some embodiments, the multivalent linker of the present disclosure comprises a plurality of peptide binding arms. In some embodiments, the peptide binding arm comprises at least one sequence from SEQ ID NO: 1-418. In some embodiments, the multivalent linker comprises two or more peptide binding arms having a sequence selected from SEQ ID NO: 1-418.

[0286] In some embodiments, the peptide binding arm is a single domain antibody or an antigen binding fragment thereof. A single domain antibody is an antibody whose complementary determining region is a part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, cattle, and / or alpacas.

[0287] According to the present disclosure, single domain antibodies as used herein are derived from naturally occurring antibodies known as heavy chain antibodies lacking light chains. Variable domains derived from naturally heavy chain antibodies lacking light chains are referred to herein as VHH molecules to distinguish them from conventional VHs of four-chain immunoglobulins.

[0288] In some embodiments, the VHH molecule may be derived from an antibody produced in a species of the family Camelidae (eg, camel, dromedary, llama, vicuna, alpaca, and guanaco).

[0289] Other species besides Camelidae can produce heavy chain antibodies that naturally lack light chains. For example, sharks produce heavy chain antibodies (commonly referred to as IgNARs) that naturally lack light chains, which contain VNAR domains. In addition, single domain antibodies (sdAbs) can be obtained from synthetic libraries. Designed ankyrin repeat proteins (DARPins) are antibody mimetic proteins that can also be generated from synthetic libraries. Other examples may include, but are not limited to, anticalins (lipocalins), single chain variable fragments (scFvs), fibronectin type III (FN3) domains, monobodies, and / or affibodies. All such sdAbs, DARPins, anticalins, scFvs, FN3 domains, monobodies, and affibodies are within the scope of the present disclosure.

[0290] In some embodiments, the peptide binding arm of the present disclosure comprises sequence identity to any one of SEQ ID NOs. 1-494.

[0291] In some embodiments, the multivalent sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs. 1-494.

[0292] In some embodiments, the multivalent sequence comprises a sequence that is between 80%-85%, between 85%-90%, between 90%-95%, or between 95%-100% identical to any one of SEQ ID NOs. 1-494.

[0293] In some embodiments, the C-terminus of the first peptide selected from SEQ ID NO: 1-418 is conjugated to the N-terminus of the second peptide via a linker segment to generate a multivalent linker. In some embodiments, the linker segment comprises the sequence GGGGS or G4S, or any other linker segment disclosed in the present disclosure.

[0294] In some embodiments, the multivalent linker comprises a plurality of G4S linker segments. For example, the multivalent linker may comprise (G4S)n, wherein n=1-10.

[0295] In some embodiments, the first peptide and the second peptide have different sequences (eg, comprise different SEQ ID NOs disclosed herein).

[0296] In some embodiments, the first peptide and the second peptide have the same sequence (eg, are copies of any of the same SEQ ID NO. sequences disclosed herein).

[0297] In some embodiments, the peptide binding arm non-covalently binds to an epitope of the target antigenic unit. In some embodiments, the peptide binding arm covalently binds to an epitope of the target antigenic unit. Molecular complex

[0298] In one aspect, the disclosure provides a molecular complex comprising i) a single target antigen unit and ii) a multivalent linker of the disclosure. In some embodiments, the molecular complex comprises a single multivalent linker, such as those of the disclosure.

[0299] In some embodiments, a single target antigen unit comprises at least two identical or substantially identical epitopes of a peptide binding arm of a multivalent linker (e.g., a multivalent linker having at least two identical or substantially identical peptide binding arms).

[0300] In some embodiments, a single target antigen unit comprises at least two different epitopes for a peptide binding arm of a multivalent linker (eg, a multivalent linker having at least two different peptide binding arms).

[0301] In some embodiments, the number of epitopes contained in a single target antigen unit is the same as the number of peptide binding arms in the multivalent linker. In some embodiments, the number of epitopes / peptide binding arms is two. In some embodiments, the number of epitopes / peptide binding arms is three. In some embodiments, the number of epitopes / peptide binding arms is four. In some embodiments, the number of epitopes / peptide binding arms is five. In some embodiments, the number of epitopes / peptide binding arms is six, seven, eight, nine, ten or more than ten.

[0302] In some embodiments, the multivalent linker is less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate from a single target antigen unit. In some embodiments, the multivalent linker dissociates at a rate of 10 -4 ,10 -5 ,10 -6 ,10 -7 ,10 -8 ,10 -9 ,10 -10 ,or10 -11 Smaller apparent k off (s -1 ) rate from a single target antigen unit. In some embodiments, the multivalent linker dissociates at a rate of less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate from a single target antigen unit. In some embodiments, the multivalent linker dissociates at a rate of less than or equal to 1.0×10 -6 The apparent k off (s -1) rate from a single target antigen unit. In some embodiments, the multivalent linker dissociates at a rate of less than or equal to 1.0×10 -7 The apparent k off (s -1 ) rate from a single target antigen unit. In some embodiments, the multivalent linker dissociates at a rate of less than or equal to 1.0×10 -8 The apparent k off (s -1 ) rate of dissociation from a single target antigen unit. In some embodiments, k off (s -1 ) rates were determined by biolayer interferometry (BLI).

[0303] In one aspect, the present disclosure provides a population of molecular complexes. In some embodiments, the present disclosure provides a composition comprising at least 1 g, at least 1 mg, at least 1 ug, or at least 1 ng of a population of molecular complexes. In some embodiments, the present disclosure provides a composition comprising at least 1 ng of a population of molecular complexes. In some embodiments, in a composition comprising a population of molecular complexes, less than 5%, 4%, 3%, 2%, or 1% of the multiple peptide binding arms of a multivalent linker are cross-linked with different target antigen units. Target antigen unit

[0304] In some embodiments, the multivalent linker is associated with a single target antigen unit. In some embodiments, a single target antigen unit and the multivalent linker are contained within a molecular complex.

[0305] In some embodiments, the target antigen unit comprises the constant region of an antibody. In some embodiments, the single target antigen unit comprises the Fc region of an antibody. In some embodiments, the epitope is located on the CH2 domain of the antibody constant region. In some embodiments, the epitope is located on the CH3 domain of the antibody constant region. In some embodiments, the epitope is located on the CH4 domain of the antibody constant region. In some embodiments, the number of epitopes is two within a single target antigen unit.

[0306] In some embodiments, the epitope is located in a position that is not in the Fc region of the antibody. In some embodiments, the epitope is located in the Fab region of the antibody. In some embodiments, the epitope is located in the CH1 domain of the antibody. In some embodiments, the epitope is located in the CL domain (of the light chain) of the antibody.

[0307] In some embodiments, the target antigen unit comprises or consists of: antibody, F(ab')2, Fab2, Fab3 or IgNAR. In some embodiments, the target antigen unit comprises or consists of: antibody. In some embodiments, the target antigen unit comprises or consists of: F(ab')2. In some embodiments, the target antigen unit comprises or consists of: Fab2. In some embodiments, the target antigen unit comprises or consists of: Fab3. In some embodiments, the target antigen unit comprises or consists of: IgNAR. In some embodiments, the target antigen unit comprises an antigen-binding fragment of an antibody.

[0308] In some embodiments, the target antigen unit comprises or consists of: scFv.

[0309] In some embodiments, the antibody is IgG. In some embodiments, the IgG antibody is IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments, the IgG antibody is IgG1 subclass. In some embodiments, the IgG antibody is IgG2 subclass. In some embodiments, the IgG antibody is IgG3 subclass. In some embodiments, the IgG antibody is IgG4 subclass.

[0310] In some embodiments, the IgG antibody is a human antibody. In some embodiments, the human IgG antibody is an IgG1, IgG2, IgG3 or IgG4 subclass. In some embodiments, the human IgG antibody is an IgG1 subclass. In some embodiments, the human IgG antibody is an IgG2 subclass. In some embodiments, the human IgG antibody is an IgG3 subclass. In some embodiments, the human IgG antibody is an IgG4 subclass. In some embodiments, the constant region of the IgG1 antibody comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 496 (Uniprot ID: P01857). In some embodiments, the constant region of the IgG2 antibody comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 497 (Uniprot ID: P01859). In some embodiments, the constant region of an IgG3 antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 498 (Uniprot ID: P01860). In some embodiments, the constant region of an IgG4 antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 499 (Uniprot ID: P01861).

[0311] In some embodiments, the IgG antibody is a mouse antibody. In some embodiments, the mouse IgG antibody is an IgG1, IgG2a, IgG2b, IgG2c or IgG3 subclass. In some embodiments, the mouse IgG antibody is an IgG1 subclass. In some embodiments, the mouse IgG antibody is an IgG2a subclass. In some embodiments, the mouse IgG antibody is an IgG2b subclass. In some embodiments, the mouse IgG antibody is an IgG2c subclass. In some embodiments, the mouse IgG antibody is an IgG3 subclass. In some embodiments, the mouse IgG1 antibody is an IgG1* variant. In some embodiments, the mouse IgG2b antibody is an IgG2b* variant. In some embodiments, the mouse IgG2a antibody is an IgG2a A allotype (IgG2aA). In some embodiments, the mouse IgG2a antibody is an IgG2a B allotype (IgG2aB). The description of mouse antibodies can be found in, for example, Han et al., ACS Omega. April 21, 2020; 5(15): 8564-8571, the contents of which are incorporated by reference in their entirety for all purposes. In some embodiments, the constant region of an IgG1 antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 500 (Uniprot ID: P01868). In some embodiments, the constant region of an IgG2a antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 501 (Uniprot ID: P01864). In some embodiments, the constant region of an IgG2b antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 502 (Uniprot ID: P01867). In some embodiments, the constant region of an IgG2c antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 503 (Uniprot ID: A0A0A6YY53). In some embodiments, the constant region of an IgG3 antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 504 (Uniprot ID: A0A075B5P5).

[0312] In some embodiments, the IgG antibody is a rat antibody. In some embodiments, the rat IgG antibody is an IgG1, IgG2a, IgG2b or IgG2c subclass. In some embodiments, the rat IgG antibody is an IgG1 subclass. In some embodiments, the rat IgG antibody is an IgG2a subclass. In some embodiments, the rat IgG antibody is an IgG2b subclass. In some embodiments, the rat IgG antibody is an IgG2c subclass. A description of rat antibodies can be found in, for example, Kinoshita and Ross, J Immunoassay. September 1993; 14 (3): 149-66, the contents of which are incorporated by reference in their entirety for all purposes. In some embodiments, the constant region of the IgG1 antibody comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 505 (UniprotID: P20759). In some embodiments, the constant region of an IgG2a antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 506 (Uniprot ID: P20760). In some embodiments, the constant region of an IgG2b antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 507 (Uniprot ID: P20761). In some embodiments, the constant region of an IgG2c antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 508 (Uniprot ID: P20762).

[0313] In some embodiments, the IgG antibody is a rabbit antibody.In some embodiments, the constant region of the IgG antibody comprises or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 509 (Uniprot ID: P01870).

[0314] In some embodiments, the antibody is IgM. In some embodiments, the antibody is IgA. In some embodiments, the antibody is IgD. In some embodiments, the antibody is IgE.

[0315] In some embodiments, the antibody is a chicken antibody.In some embodiments, the antibody is an IgY antibody.

[0316] In some embodiments, the antibody is a heavy chain antibody.

[0317] In some embodiments, the antibody is a mouse antibody, a rat antibody, a rabbit antibody, a chicken antibody (e.g., IgY), a guinea pig antibody, a donkey antibody, a human antibody, a goat antibody, a pig antibody, a horse antibody, or a cow antibody. In some embodiments, the antibody is a mouse antibody. In some embodiments, the antibody is a rat antibody. In some embodiments, the antibody is a rabbit antibody. In some embodiments, the antibody is a chicken antibody. Generation of multivalent linkers

[0318] The present disclosure teaches methods for producing multivalent linkers. In some embodiments, the multivalent linkers of the present disclosure include a general structure: (peptide binding arm)-(linker segment)-(peptide binding arm). In some embodiments, the linker segment is a polypeptide. Therefore, in some embodiments, the multivalent linker is a polypeptide that can be encoded by a nucleic acid and expressed from a nucleic acid. Therefore, in some embodiments, the multivalent linker of the present disclosure is prepared by in vivo or in vitro expression (e.g., translation). Those skilled in the art will be familiar with the technology of producing translation fusions (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, 1992) New York, which is incorporated herein by reference in its entirety).

[0319] Briefly, in some embodiments, a vector is designed that expresses a polypeptide in a linear and in-frame manner, the polypeptide comprising [a peptide binding arm selected from SEQ ID NO: 1-418]-[an amino acid linker segment as described herein]-[a peptide binding arm selected from SEQ ID NO: 1-418]. In some embodiments, the multiple peptide binding arms can be the same. In some embodiments, the multiple peptide binding arms can be different. How to use

[0320] The multivalent linkers of the present disclosure can generally be used in any method requiring the attachment of a compound to an antibody or other epitope.

[0321] For example, the method may include detecting the target antigen by optical detection, isotope detection, or electron microscopy detection. For example, the method of the present disclosure may be a microscopy method, such as a fluorescence microscopy method, or an immunofluorescence method.

[0322] The methods of the present disclosure may also include immunofluorescence detection. In some embodiments, the method includes cyclic immunofluorescence detection.

[0323] The methods of the present disclosure may also include spatial genomic analysis.

[0324] The methods of the present disclosure may also include flow cytometry or fluorescence activated cell sorting (FACS) methods. The methods of the present disclosure may also include Western blotting.

[0325] The method of the present disclosure may also be an immunohistochemical method. The method of the present disclosure may also be an ELISA method.

[0326] In some embodiments, the methods of the present disclosure encompass contacting a second monovalent antibody with a first antibody (eg, when performing an ELISA).

[0327] The method of the present disclosure may also include screening antibodies or molecules comprising antigen binding fragments thereof. In some embodiments, the method includes screening of hybridomas. In some embodiments, the method includes labeling the supernatant (e.g., hybridoma supernatant) with a multivalent linker of the present disclosure.

[0328] The methods of the present disclosure may also include mass spectrometry analysis.

[0329] In some embodiments, the method includes detecting a test antigen in a sample. In some embodiments, the method includes the following steps: A) contacting the sample with a multivalent linker and a binding agent capable of specifically binding to a test antigen, B) removing unbound binder and multivalent linker, and C) Measuring the presence of the multivalent linker in the sample.

[0330] In some embodiments, the multivalent linker forms a molecular complex with the binding agent via the target antigen unit of the latter.

[0331] In some embodiments, the method comprises detecting one or more test antigens in a sample, comprising contacting the sample with one or more of said molecular complexes, wherein the molecular complex is formed by a single binding agent and a corresponding multivalent linker, wherein the binding agent is capable of specifically binding to the test antigen. In some embodiments, the method comprises detecting at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 test antigens.

[0332] In some embodiments, the method includes detecting two or more test antigens. In some embodiments, the method includes contacting the sample with a first binding agent specific for the first test antigen, a second binding agent specific for the second test antigen, optionally a third binding agent specific for the third test antigen, and the like, and adding a multivalent linker that can be specifically bound to each binding agent, respectively. In some embodiments, the multivalent linker can be added before, at the same time, or after the corresponding binding agent. In some embodiments, the method includes contacting the sample with a first molecular complex specific for the first test antigen and a second molecular complex specific for the second test antigen, and optionally a third molecular complex for the third test antigen, etc. In some embodiments, the method includes contacting the sample with a mixture of molecular complexes and binding agents, and then adding a multivalent linker corresponding to the binding agent. Therefore, in some embodiments, the first test antigen can be detected by directly adding the corresponding molecular complex to the sample, and the second test antigen can be detected by adding the binding agent and the corresponding multivalent linker to the sample separately, and vice versa.

[0333] In some embodiments, less than 5%, 4%, 3%, 2%, or 1% of the multivalent linkers are bound to two or more of the binding agents (cross-linked to two or more binding agents).

[0334] In some embodiments, less than 5%, 4%, 3%, 2% or 1% of the first multivalent linker is bound to the second binding agent, and at the same time less than 5%, 4%, 3%, 2% or 1% of the second multivalent linker is bound to the first binding agent. In some embodiments, less than 5%, 4%, 3%, 2% or 1% of each type of different multivalent linker is bound to a non-target binding agent. In some embodiments, for each type of different multivalent linker, the ratio is less than 1%.

[0335] In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent comprises an antigen-binding fragment of an antibody.

[0336] In some embodiments, the two or more binding agents comprise the same target antigen unit (or epitope) to which the same multivalent linker specifically binds, but the binding agents are added to the sample after being premixed with the multivalent linker with a unique reporter, and due to the slow dissociation rate, the method prevents or minimizes the binding of each multivalent linker with a unique reporter to non-target binding agents within the time frame of the method.

[0337] In some embodiments, the method of the present disclosure needs to quench unbound multivalent linkers so that they are not bound to undesirable targets. In some embodiments, quenching is achieved by adding a bait molecule (i.e., a quencher) that can be bound to a multivalent linker. In some embodiments, the bait molecule comprises an epitope or target antigen unit identified by a multivalent linker, but the bait molecule lacks the ability to bind to any test antigen. In some embodiments, the bait molecule is a non-specific antibody or a fragment thereof (e.g., an Fc fragment). In some embodiments, the bait molecule is an Fc fragment. In some embodiments, the non-specific antibody is an IgG. In some embodiments, the non-specific antibody is polyclonal. In some embodiments, the non-specific antibody is monoclonal.

[0338] In some embodiments, a decoy molecule may contain a tag or moiety that facilitates its own removal (along with excess multivalent linkers).

[0339] In some embodiments, the method includes removing unbound multivalent linkers from the multivalent linker-binder complex.

[0340] In some embodiments, the unbound multivalent linker is removed by ultrafiltration. In some embodiments, the unbound multivalent linker is removed by bead depletion. Therapeutic agents

[0341] In some embodiments, the multivalent linker of the present disclosure can be labeled with cargo including, but not limited to, fluorescent dyes, haptens (e.g., biotin), contrast agents (e.g., gadolinium, radionuclides), chelated metals, therapeutic agents, sensitizers, small molecules, or combinations thereof. In some embodiments, the therapeutic agent is attached to the multivalent linker.

[0342] In some embodiments, the therapeutic agent is a chemotherapeutic agent, a therapeutic antibody, a nucleic acid molecule, a radioisotope, a thymidylate synthase inhibitor, or a platinum compound. Illustrative examples of each are found in US Pat. No. 10,888,618, which is incorporated by reference in its entirety. Buffer

[0343] In some embodiments, the multivalent linker is prepared in a pharmaceutically acceptable carrier, buffer or excipient that is non-toxic and / or stabilizes the multivalent linker. In some embodiments, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN TM ) Polyethylene glycol (PEG) and poloxamer (PLURONICS TM )wait. Reporter and / or effector molecules

[0344] In some embodiments, the reporter molecule is attached to a multivalent linker. Substances suitable for attachment to a multivalent linker include, but are not limited to, amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymer microparticles, biological cells, viruses, fluorophores, chromophores, dyes, toxins, haptens, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semisolid matrices, and combinations thereof. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, and are incorporated herein by reference in their entirety.

[0345] In some embodiments, the multivalent linker of the present disclosure is labeled with a radioactive isotope, including but not limited to radioactive isotopes of At211, Cu64, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Zr89, and Lu.

[0346] In some embodiments, the reporter molecule is connected to the multivalent linker on the gene. For example, the reporter molecule can be a fluorescent molecule, such as green fluorescent protein (GFP). In order to connect on the gene, the sequence of GFP will be in the same frame as the sequence of the multivalent linker. The expression or transcription of the coding sequence can be affected or controlled by the same promoter. Therefore, expression from the promoter will express the reporter molecule and the multivalent linker in the same polypeptide.

[0347] In some embodiments, the reporter is located at the C-terminus of the multivalent linker. In some embodiments, the sequence encoding the multivalent linker does not include a stop codon at the end of the peptide binding arm, but includes a stop codon after the sequence encoding the reporter.

[0348] In some embodiments, the reporter molecule is located at the n-terminus of the multivalent linker.

[0349] In some embodiments, the reporter / effector molecules are attached by other means. For example, in some embodiments, the reporter and effector can be attached by photoactivated site-specific conjugation of native IgG (Bioconjugate Chem. 2015, 26, 8, 1456-1460). Fluorescent reporter

[0350] In some embodiments, the multivalent peptide is conjugated to a fluorescent reporter. In some embodiments, fluorescent reporters include, but are not limited to, coumarins, cyanines, benzofurans, quinolines, quinazolinones, indoles, benzoxazoles, borapolyazaindacenes, and xanthenes (including fluorescein, rhodamine, and rhodol).

[0351] In some embodiments, the fluorescent reporter is green fluorescent protein (GFP). GFP refers to a polypeptide having a peak at or about 510 nm in the emission spectrum. A variety of fluorescent proteins (FPs) emitting at different wavelengths are known in the art. The FP of interest is green fluorescent protein (GFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), far-red (including but not limited to) fluorescent protein or near-infrared fluorescent protein. As used herein, Aequorea GFP refers to GFP from Aequorea and its mutants or variants. Such mutants and others such as Anthozoa reef coral, Anemonia sea anemone, Renilla, Galaxea coral, Acropora brown coral, Trachyphyllia, and peti GFP (from Pectimidae corals) and from other species are well known and available and known to those skilled in the art. Additional GFP variants include, but are not limited to, BFP, CFP, YFP, and OFP.Examples of fluorescent proteins and their variants include: GFP proteins, such as Emerald (Invitrogen, Carlsbad, California), EGFP (Clontech, Palo Alto, California), Azami-Green (MBL International, Woburn, Massachusetts), Kaede (MBL International, Woburn, Massachusetts), ZsGreenl (Clontech, Palo Alto, California) and CopGFP (Evrogen / Axxora, LLC, San Diego, California); CFP proteins, such as Cerulean (Rizzo, Nat Biotechnol. 22(4):445-9 (2004)), mCFP (Wang et al., PNAS USA. 101(48):16745-9 (2004)), AmCyan1 (Clontech, Palo Alto, California), MiCy (MBL International, Woburn, Massachusetts) and CyPet (Nguyen and Daugherty, Nat Biotechnol. 22(4):445-9 (2004)). Biotechnol. 23(3):355-60 (2005)); BFP proteins such as EBFP (Clontech, Palo Alto, CA); YFP proteins such as EYFP (Clontech, Palo Alto, CA), Ypet (Nguyen and Daugherty, Nat Biotechnol. 23(3):355-60 (2005)), Venus (Nagai et al., Nat. Biotechnol. 20(1):87-90 (2002)), Zs yellow (Clontech, Palo Alto, CA), and mCitrine (Wang et al., PNAS USA. 101(48):16745-9 (2004)); OFP proteins such as cOFP (Strategene, La Jolla, CA), mKO (MBL International, Woburn, MA), and mOrange; et al. (Shaner NC, Steinbach PA, and Tsien RY., Nat Biotechnol. 23(3):355-60 (2005)), Venus (Nagai et al., Nat ... Methods. 2(12):905-9(2005)).

[0352] In some embodiments, the fluorescent reporter is a red fluorescent protein (RFP). In some embodiments, the RFP is a DsRed (Matz et al., Nature Biotechnology 17:969-973 (1999)) isolated from the corallimorph Discosoma and any other optional species, such as red fluorescent protein or far-red fluorescent protein from Heteractis reef-building corals and Actinia or Entacmaea anemones and variants thereof. RFPs include monomeric red fluorescent protein 1 (mRFP1), mCherry, tdTomato, mStrawberry, mTangerine (Wang et al., PNAS USA. 101(48):16745-9 (2004)), DsRed2 (Clontech, Palo Alto, California), and DsRed-T1 (Bevis and Glick, Nat. Biotechnol., 20:83-87 (2002)), Anthomedusa J-Red (Evrogen), and Aspleniformis AsRed2 (Clontech, Palo Alto, California), including discosome variants. Far-red fluorescent proteins include, for example, Anemone AQ143 (Shkrob et al., Biochem J. 392(Pt 3):649-54 (2005)), Anemone eqFP611 (Wiedenmann et al. Proc Natl Acad Sci USA. 99(18):1 1646-51 (2002)), discoid variants such as mPlum and mRasberry (Wang et al., PNAS USA. 101(48):16745-9 (2004)), and Anemone HcRed1 and t-HcRed (Clontech, Palo Alto, California). Non-fluorescent reporter

[0353] As used herein, the term "non-fluorescent reporter" refers to a chemical moiety that does not fluoresce but can be used to provide contrast or signal in imaging and can be detected by non-fluorescent imaging techniques. In certain embodiments, other non-fluorescent reporters can be chemically linked to the imaging agent or can be administered to the subject simultaneously or sequentially with the imaging agent of the present disclosure. Such reporters can include photoluminescent nanoparticles, radioisotopes, superparamagnetic agents, X-ray contrast agents, and ultrasound agents. Reporters can also include therapeutic reporters, such as porphyrins, Aminolevulinic acid, hypericin, benzoporphyrin derivatives, and radionuclides used in radiotherapy. Enzyme reporter

[0354] The reporter molecule may include one or more enzyme reporters. In some embodiments, the reporter is horseradish peroxidase. In some embodiments, the reporter is beta-galactosidase or alkaline phosphatase. In some embodiments, the enzyme reporter is acetylcholinesterase. In some embodiments, the enzyme reporter is a binding pair capable of forming a complex. In some embodiments, the binding pair is streptavidin and biotin. In some embodiments, the binding pair is avidin and biotin. Other enzyme reporters are well known in the art and are further described in van Rossum et al., which is incorporated by reference in its entirety (van Rossum, T., Kengen, SWM and van der Oost, J. (2013), Reporter-based screening and selection of enzymes. FEBS J, 280: 2979-2996.) Radioactive Reporter

[0355] The reporter molecule may include one or more radioactive labels. Radioisotopic forms of metals such as copper, gallium, indium, technetium, yttrium, and lutetium may be chemically linked to a multivalent linker. Exemplary radioactive labels include, but are not limited to 3 H. 35 S. 14 C. 32 P. 99m TC, 111 In, 64 Cu, 67 Ga, 186 Re, 188 Re, 153 Sm, 177 Lu and 67 Cu.

[0356] Additional markers may include, for example 123 I. 124 I. 125 I. 11 C. 13 N. 15 O and 18 F. Additional labels may be therapeutic radiopharmaceuticals, including for example 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm,90 Y. 212 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 67 Cu, 105 Rh, 111 Ag and 192 Ir.

[0357] The chelating agent or bonding moiety can be chemically associated with the multivalent linker. The chelating agent can be selected to bind to a radioisotope (e.g., 99m Tc, 111 In, 64 Cu and 67 Ga) to form a stable complex. Exemplary chelating agents include diamine dithiols, monoamine-monoamide dithiols, triamide-monothiols, monoamine-diamide-monothiols, diamine dioximes, and hydrazines. Chelating agents are typically tetradentate with donor atoms selected from nitrogen, oxygen and sulfur and may include, for example, cyclic and non-cyclic polyaminocarboxylates such as diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), (DO3A), 2-benzyl-DOTA, α-(2-phenylethyl)1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA and 6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl]-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

[0358] The chelating agent or bonding moiety can be selected to bind to a radioactive isotope (e.g., 186 Re, 188 Re, 153 Sm, 177 Lu and 67The chelating agent can be selected from diamine dithiols, monoamine-monoamide dithiols, triamide-monothiols, monoamine-diamide-monothiols, diamine dioximes and hydrazines, cyclic and non-cyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA, α-(2-phenylethyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA and 6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl]-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine. Metal Reporter / Effector

[0359] In some embodiments, the reporter molecule may include one or more magnetic labels. Other exemplary reporters may include chelators for magnetic reagents. Such chelators may include, for example, polyamine-polycarboxylate chelators or iminoacetic acid chelators that may be chemically linked to a multivalent linker.

[0360] Magnetic reporters can be selected to form stable complexes with paramagnetic metal ions such as Gd(III), Dy(III), Fe(III) and Mn(II) and are selected from cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA, α-(2-phenylethyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA and 6,6"-bis[N,N,N",N"-tetra(carboxymethyl)aminomethyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine.

[0361] In some embodiments, multivalent linkers are chemically attached to superparamagnetic metal oxide nanoparticles that are (a) non-fluorescent, or (b) fluorescent, and can be used in a variety of in vitro and in vivo applications. Oligonucleotide reporter

[0362] In some embodiments, the multivalent linker of the present disclosure comprises one or more oligonucleotide reporters. Those skilled in the art will also be familiar with various natural and synthetic oligonucleotides that can be attached to the antibodies or multivalent linkers of the present disclosure, including those discussed in the following: Evaluation of oligonucleotide conjugated antibodies as reporter molecules in single-cell assays, Takahashi et al., The Journal of Immunology May 1, 2020, 204 (Supplement 1) 86.35; Pharmaceutics. June 2020; 12 (6): 545. Published online on June 12, 2020. Doi: 10.3390 / pharmaceutics12060545; and Kennedy-Darling et al., (2021), Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. Eur. J. Immunol., 51: 1262-1277. Compositions comprising multivalent linkers

[0363] In some embodiments, the multivalent linker or molecular complex is prepared in a pharmaceutically acceptable carrier, buffer or excipient that is non-toxic and / or stabilizes the multivalent linker or molecular complex. In some embodiments, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN TM ) Polyethylene glycol (PEG) and poloxamer (PLURONICS TM )wait.

[0364] In some embodiments, the composition can be used in a form suitable for any route of administration, including, for example, oral administration in the form of tablets, capsules or liquids, or injection in the form of sterile aqueous solutions. The composition can be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders prepared with water or other suitable vehicles before use, and these forms optionally have flavorings and coloring agents for immediate release, delayed release, modified release, sustained release, pulse release or controlled release applications. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powders, pastes, granules, bullets or premixed preparations can also be used. Solid and liquid compositions for oral use can be prepared according to methods well known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients, which can be in solid or liquid form. When the composition is formulated for oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as: binders (e.g., pregelatinized starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

[0365] Pharmaceutically acceptable excipients also include, but are not limited to, microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate and glycine, disintegrants such as starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate, cross-linked sodium carboxymethylcellulose and certain complex silicates, and granulation binders such as polyvinyl pyrrolidone, hydroxypropyl ethylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and gum arabic. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0366] In some embodiments, a composition comprises a multivalent linker and a binding agent (eg, a primary antibody).

[0367] In some embodiments, the composition comprises a first multivalent linker and a first binding agent (e.g., a primary antibody). In some embodiments, the composition comprises a second multivalent linker and a second binding agent (e.g., a primary antibody). In some embodiments, the first binding agent and the second binding agent (e.g., a primary antibody) are from the same species. In some embodiments, the first binding agent and the second binding agent (e.g., a primary antibody) are from different species.

[0368] In some embodiments, a composition comprises a multivalent linker, a binding agent (eg, a primary antibody), and an antigen.

[0369] In some embodiments, the composition comprises a first multivalent linker, a first binding agent (e.g., a primary antibody) and a first antigen. In some embodiments, the composition comprises a second multivalent linker, a second binding agent (e.g., a second primary antibody) and a second antigen. In some embodiments, the first binding agent and the second binding agent (e.g., a primary antibody) are from the same species. In some embodiments, the first binding agent and the second binding agent (e.g., a primary antibody) are from different species.

[0370] In some embodiments, the binding agent is or comprises a target antigen unit as described in the present disclosure. Steps for labeling with multivalent linkers

[0371] In some embodiments, the present disclosure teaches the step of labeling the multivalent linker with a primary binding agent (e.g., a primary antibody) prior to conducting an experimental assay. In some embodiments, labeling allows the use of primary binding agents from the same species or with a common epitope.

[0372] In some embodiments, a multivalent linker labels a primary antibody. In some embodiments, a primary antibody is a monoclonal antibody. In some embodiments, the antibody is not a polyclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0373] In some embodiments, the primary antibody and the multivalent linker are mixed at a specific molar ratio.

[0374] In some embodiments, the molar ratio of the primary binding agent (e.g., primary antibody) to the multivalent linker is about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or 1:10.

[0375] In some embodiments, the molar ratio of the primary binding agent (e.g., primary antibody) to the multivalent linker is between 1:1-1:1.5, 1:1.5-1:2, 1:2-1:2.5, 1:2.5-1:3, 1:3-1:3.5, 1:3.5-1:4, 1:4-1:4.5, 1:4.5-1:5, 1:5-1:5.5, 1:5.5-1:6, 1:6-1:6.5, 1:6.5-1.7, 1:7-1:7.5, 1:7.5-1:8, 1:8-1:8.5, 1:8.5-1:9, 1:9-1:9.5, or 1:9.5-1:10.

[0376] In some embodiments, the concentration of the primary binding agent (eg, primary antibody) should be maintained at a high concentration during the pre-conjugation step.

[0377] In some embodiments, the concentration of the primary antibody is at least 0.00001 g / l, at least 0.0001 g / l, at least 0.001 g / l, at least 0.01 g / l, at least 0.05 g / l, at least 0.1 g / l, at least 0.15 g / l, at least 0.2 g / l, at least 0.25 g / l, at least 0.3 g / l, at least 0.35 g / l, at least 0.4 g / l, at least 0.45 g / l, at least 0.5 g / l, at least 0.55 g / l, at least 0.6 g / l, at least 0.65 g / l, at least 0.7 g / l, at least 0.75 g / l, at least 0.8 g / l, at least 0.85 g / l, at least 0.9 g / l, at least 0.95 g / l, or at least 1 g / l.

[0378] In some embodiments, the concentration of the primary antibody is between 0.00001 g / l and 0.0001 g / l, between 0.0001 g / l and 0.001 g / l, between 0.001 g / l and 0.01 g / l, between 0.01 g / l and 0.05 g / l, between 0.05 g / l and 0.1 g / l, between 0.1 g / l and 0.15 g / l, between 0.15 g / l and 0.2 g / l, between 0.2 g / l and 0.25 g / l, between 0.25 g / l and 0.3 g / l, between 0.3 g / l and 0.35 g / l, between 0.35 g / l and 0.4 g / l, 1 g / l, 0.4 g / l to 0.45 g / l, 0.45 g / l to 0.5 g / l, 0.5 g / l to 0.55 g / l, 0.55 g / l to 0.6 g / l, 0.6 g / l to 0.65 g / l, 0.65 g / l to 0.7 g / l, 0.7 g / l to 0.75 g / l, 0.75 g / l to 0.8 g / l, 0.8 g / l to 0.85 g / l, 0.85 g / l to 0.9 g / l, 0.9 g / l to 0.95 g / l, or 0.95 g / l to 1 g / l, including all ranges and subranges therebetween.

[0379] In some embodiments, the binding agent (e.g., a primary antibody) is incubated with the multivalent linker for at least 1 second, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 120 minutes, at least 180 minutes, at least 240 minutes, or at least 300 minutes.

[0380] In some embodiments, the binding agent (e.g., a primary antibody) is incubated with the multivalent linker for at most 1 second, at most 1 minute, at most 5 minutes, at most 10 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 120 minutes, at most 180 minutes, at most 240 minutes, or at most 300 minutes.

[0381] In some embodiments, the binding agent (e.g., a primary antibody) is incubated with the multivalent linker for between 1 second and 1 minute, between 1 minute and 5 minutes, between 5 minutes and 10 minutes, between 10 minutes and 20 minutes, between 20 minutes and 30 minutes, between 30 minutes and 40 minutes, between 40 minutes and 50 minutes, between 50 minutes and 60 minutes, between 60 minutes and 120 minutes, between 120 minutes and 180 minutes, between 180 minutes and 240 minutes, or between 240 minutes and 300 minutes, including all ranges and subranges therebetween.

[0382] In some embodiments, the glycerol content is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. Example Example 1: Synthesis of phage display libraries

[0383] As noted above, in some embodiments, the peptide binding arm may be derived from an antibody (e.g., a VHH or a single domain antibody (sdAb)). The peptide binding arm of the present disclosure can be generated by immunizing a mammal (including a camelid or a rodent) with a target antigen. Depending on the antigen, peptide binding arms with even picomolar affinities may be identified without additional in vitro maturation steps. The binding region of the identified antibody (e.g., from a camel) may then be used as a peptide binding arm in the methods and compositions of the present disclosure.

[0384] Since the 1990s, the method of separating antigen-specific antibodies from immune antibody libraries by phage display and other display technologies is well known, well established, and these schemes are used routinely. In order to generate immune libraries, mammals (such as camelids or rodents) will be immunized with a limited amount of target antigens within a certain period of time. After the immune stage, B cells of secretory antigens will be isolated from peripheral blood, total mRNA will be extracted and transcribed into cDNA. Subsequently, multiple PCR steps will be carried out to amplify binding variants, and large and diverse phage libraries representing complete immune antibody libraries will be generated (Pleiner et al., 2015). Example 2: Phage display panning and screening process

[0385] The phage display screening method described above was used to provide a high-throughput and unbiased method to identify peptide binding arms for use in multivalent linkers.

[0386] Phage display assays were performed according to methods known in the art and further as described in Pande et al. and Wu et al. (Pande, J et al. Phage display: Concept, innovations, applications and future. Biotechnology Advances 28 (2010) 849-858; Wu, CH., Liu, IJ., Lu, RM. et al. Advancement and applications of peptide phage display technology in biomedical science. J Biomed Sci 23, 8 (2016)).

[0387] In short, phage display is based on genotype-phenotype coupling, i.e., plasmid+phage (phagemid) encodes a multivalent linker fused to one of the phage coat proteins (e.g., pIII). The expressed multivalent linker is connected to the coat protein and presented on its surface by the phage. During the panning process, multivalent linker fragments from a diverse library that bind to the target antigen are enriched and selected. In general, the selection process proceeds as follows: 1. Bind the multivalent linker-phage complex to the antigen in solution or immobilized on a plate. 2. Unbound multivalent linker-phage complexes are washed away through multiple washing steps, thereby retaining only bound antigen-binding complexes. 3. The antigen-specific candidates are then eluted, for example by a pH change. 4. The free multivalent linker-phage complexes can then be used for bacterial infection and phage amplification, which can be used as input for subsequent selection rounds to further enrich for candidates with desired properties.

[0388] After several rounds of selection, the encoded multivalent linkers are subjected to high throughput screening using monoclonal clones. For screening, single colonies are picked, the multivalent linkers are expressed by E. coli in a 96-well plate format, and the multivalent linkers are tested in bacterial lysate format, for example, in ELISA, for antigen binding. Positive so-called "primary" hits can then be further characterized in additional screening, for example, for cell binding, cross-reactivity, or binding strength.

[0389] To further characterize promising candidates, the multivalent linker is expressed and purified. For example, the exact affinity can be determined with the purified candidate and final characterization can be performed in a variety of assays and applications.

[0390] The screen successfully identified hundreds of candidates (see, e.g., SEQ ID NOs: 1-418), which were further characterized. Example 3: Generation of multivalent linkers

[0391] Nucleotide sequences from the above phage display libraries were used to clone, express and purify peptide binding arms for use in multivalent linkers.

[0392] Cloning, protein expression and protein purification were performed according to methods known in the art and further described in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, 1992) New York.

[0393] The nucleotides encoding the protein binding head and peptide linker segments (e.g., from SEQ ID NO: 1-418) produced in Examples 1 and 2 are cloned into mammalian expression vectors and transfected into mammalian cells. Stable or transient transfection into mammalian cells is suitable. Alternatively, the nucleotides can be cloned into bacterial expression vectors. Stable and transient transfection into bacterial cells and / or yeast cells or any other system is suitable.

[0394] Mammalian cells were incubated at 37°C, 5% CO2 for several days, including daily media changes.

[0395] The supernatant containing the multivalent linker is purified by affinity chromatography. In addition, other methods, such as cation / anion exchange, size exclusion and / or hydrophobic interaction chromatography, are also suitable. The multivalent linker is concentrated by selective filtration and formulated in a buffer that stabilizes the free tetramer.

[0396] Dozens of multivalent proteins from Tables 1 and 2 were successfully cloned, expressed, purified, and further characterized. Example 4: Attachment of labels to multivalent linkers

[0397] The selected multivalent linker is covalently attached to the fluorophore label using standard maleimide labeling chemistry (see, e.g., Jagpreet S. Nanda, Jon R. Lorsch, Chapter 7 - Labeling of a Protein with Fluorophores Using Maleimide Derivitization).

[0398] Those skilled in the art will recognize that other labels will be compatible with the multivalent linkers of the present disclosure, including those disclosed herein and in EP 3596464 and WO 2022115791 A1, which are incorporated by reference in their entireties. Example 5: Labeling of primary IgG with multivalent linkers Initial Mark

[0399] In some embodiments, the present disclosure teaches a multivalent linker that has been bound to a primary antibody. Thus, in some embodiments, the present disclosure teaches methods of labeling a primary antibody with a multivalent linker.

[0400] For example, to use a multivalent linker with multiple primary antibodies of the same species, an initial labeling step is performed to bind each multivalent linker to the desired primary IgG. The primary IgG can be from any species. The multivalent linker disclosed in Table 1 and Table 2 was incubated with the corresponding primary IgG at a ratio of IgG: multivalent peptide = 1: 2.5. The multivalent linker was bound to the Fc region of each primary antibody. (Note that the multivalent linker can and has been targeted to other conserved regions of the primary antibody, such as the conserved light chain region.) The incubation was carried out in PBS and was protected from light at room temperature under shaking (700 rpm) for 0.5 h. In general, during the pre-conjugation step, the IgG concentration should be kept as high as possible (> 0.01 g / l). The primary antibody concentration in the specific reaction has been shown to still work at a concentration as low as 0.5 ug / ul. The reaction can be carried out with antibodies in any formulation containing glycerol or sodium azide.

[0401] Next, a multivalent linker that specifically binds to the Fc domain of the primary IgG is incubated at a ratio of IgG:multivalent linker = 1:2.5. The incubation is performed in PBS and protected from light at room temperature for 5 minutes. The primary antibody concentration in the specific reaction can be as low as 0.5ug / ul. Removal of unbound multivalent linkers

[0402] Any unbound multivalent linker may bind to non-target antigens in the immunoassay, resulting in increased background. Therefore, any unbound multivalent linker should be removed (e.g., by filtration as discussed below), depleted (as discussed below), or quenched (as discussed below).

[0403] After labeling as discussed above, unbound multivalent linkers were removed by 1) either using ultrafiltration (e.g., using Amicon Ultra 0.5 ml, 50 MWCO, Merck Millipore, catalog number UFC505024), 2) in other experiments, unbound multivalent linkers were removed by bead depletion (using agarose beads conjugated to the corresponding IgG: e.g., rabbit IgG agarose, Sigma, catalog number A2909, or mouse IgG1 Flag M2 agarose, Sigma, catalog number A2220). In other experiments, unbound multivalent linkers were quenched by adding nonspecific polyclonal IgG containing the same subtype as the primary IgG used, as discussed in more detail below. Ultrafiltration

[0404] In some cases, the primary antibody labeled with a multivalent linker is purified before packaging / use. In some cases, the labeled antibody is subjected to ultrafiltration. The labeled mixture (50-500 μl) of the primary antibody and the multivalent linker is added to the column respectively, and centrifuged at 14000g for 1min at 4°C. The eluted fraction is roughly quantified and discarded using a pipette. Then, PBS with the same volume as the discarded fraction is added to each column again. This purification step is repeated four times. After the last round (the 5th round), the column is inverted and placed in a new tube. Centrifugation is carried out at 4°C for 2min at 1000g. The eluted fraction is quantified, and finally the volume missing compared to the original volume (before the 1st round of purification) is added. Exhaustion

[0405] For bead exhaustion, the pre-conjugated mixture is processed by incubation and centrifugation of IgG-beads in a spin column for five consecutive rounds. In detail, 10 μl of IgG agarose slurry pre-balanced with PBS is respectively pipetted into a spin column. Then, the pre-conjugated mixture is added to the spin column and incubated for 2 min at room temperature under oscillation (700 rpm) on a constant temperature mixer. Further, the mixture is centrifuged (at room temperature, 1 min, 2500 g), and the eluate is used for the next round of purification. Each sample is repeated for four rounds of this purification step. Quenching

[0406] To quench unbound multivalent linkers, nonspecific IgG (e.g., mouse IgG1 isotype control monoclonal antibody or IgG control rabbit polyclonal antibody) is added to the pre-conjugated mixture. In detail, each pre-conjugated mixture is incubated for 5 min with 10 times the amount of the applied primary IgG (e.g., 10 μg nonspecific IgG for 1 μg specific primary IgG, different amounts are also expected to be feasible).

[0407] This removal and quenching step provides several methods to remove unbound multivalent linkers, which will reduce background in subsequent experiments. Example 6: Multivalent linkers of the present disclosure allow multiple labeling of primary antibodies from the same species in immunomicroscopy Leakage test

[0408] Traditional immunoassays rely on labeled secondary antibodies that bind to and label primary antibodies that target the epitope of interest. However, these secondary antibodies target common regions in primary antibodies of the same species, which limits the ability to perform multiple assays using primary antibodies from the same species. The technical problem that arises is that the secondary antibody that was previously conjugated to the first primary antibody detaches from the antibody and can then rebind to another primary antibody of the same species, which can target a different epitope in the sample (i.e., the label leaks from one primary antibody to another primary antibody of the same species). This essentially results in a label that was originally intended to label one epitope but instead labels a different epitope.

[0409] In order to demonstrate the excellent multiplexing functionality of the multivalent linkers of the present disclosure, a leakage experiment was performed that compared the signal leakage of samples labeled with a control monovalent linker with samples labeled with several multivalent linkers having different linker segment lengths. The results of this experiment are depicted in Figure 3 , and are further described below.

[0410] 5 μg of anti-ATP50 primary antibody was pre-labeled with one of the following: i) a control monovalent linker (column 1), ii) a bivalent multivalent linker with a (G4S)3 linker segment (column 2), iii) a bivalent multivalent linker with a (G4S)4 linker segment (column 3), or iv) a bivalent multivalent linker with a (G4S)5 linker segment (column 4). Each of the monovalent linkers and multivalent linkers was labeled with the AF488 fluorophore.

[0411] Selected samples also received 10 μg of unlabeled anti-LaminB1 primary antibody from the same species as the anti-ATP50 antibody. A positive control with labeled anti-laminB1 was also included in the experimental design. The incubation volume was 50 μl, corresponding to a primary dilution of 1:10. In order to remove unbound multivalent linkers, ultrafiltration was applied as described above (using a 1:20 dilution of the pre-conjugated mixture).

[0412] The experimental design of this experiment is as follows. The sample labeled as ATP50 / linker+Lamin contains: an anti-ATP50 primary antibody labeled with a corresponding monovalent or multivalent linker having an AF488 fluorophore; and an unlabeled anti-lamin primary antibody. The sample labeled as ATP50 / linker (positive control) contains only an anti-ATP50 primary antibody labeled with a corresponding monovalent or multivalent linker having an AF488 fluorophore, and does not contain other antibodies. The sample labeled as Lamin / linker (positive control) contains only an anti-Lamin primary antibody labeled with a corresponding monovalent or multivalent linker having an AF488 fluorophore, and does not contain other antibodies.

[0413] Lamin is a nuclear membrane localized protein, so samples containing fluorophore-labeled anti-Lamin will show strong nuclear fluorescence signals. (See Figure 3 ATP50 is a mitochondrial protein, so the fluorophore-labeled anti-ATP50 only shows extranuclear signals. (See Figure 3 The top sample in this experiment contains fluorophore-labeled anti-ATP50, and an unlabeled anti-Lamin primary antibody. The only way the anti-Lamin antibody can produce a fluorescent signal in the nuclear membrane is when the fluorophore label itself detaches from the anti-ATP50 and reattaches to the Lamin antibody (i.e., when the signal leaks).

[0414] By comparing the nuclear fluorescence signals of each sample, the amount of label leakage can be quantified. Specifically, the nuclear fluorescence signal from ATP50 / linker+Lamin is divided by the nuclear fluorescence signal of ATP50 / linker (positive control) to obtain the "relative nuclear signal increase". A relative nuclear signal increase of 1 indicates no signal leakage, while a value greater than 1 indicates that at least a portion of the fluorophore label from anti-ATP50 has "leaked" to the anti-Lamin antibody. The Lamin / linker (positive control) sample verifies that the anti-Lamin antibody can correctly target the nuclear membrane and fluorescently label the nuclear membrane.

[0415] like Figure 3 It can be seen that the monovalent linker exhibited significant leakage, showing a relative nuclear signal increase of 1.49 (about 49% signal leakage). Among the multivalent linkers, the (G4S)4 and (G4S)5 linker segments provided the best results, with leakage close to zero. The multivalent linker with the (G4S)3 linker segment exhibited a slightly higher background, which suggests that the linker may be too short to maintain contact with the primary antibody.

[0416] These results demonstrate that the multivalent linkers of the present disclosure are significantly superior to monovalent linkers and further show the importance of linker segment length. Example 7: Multivalent linkers allow multiple labeling of primary antibodies in Western blots

[0417] Multiplexing of multivalent linkers is not limited to immunofluorescence assays but can also be used in different types of immunoassays such as SDS-PAGE and western blot.

[0418] Those skilled in the art will be familiar with the process of preparing a Western blot. Briefly, the desired cell lysate can be separated by SDS-PAGE in advance and blotted on nitrocellulose or PVDF. On the day of the experiment, the blot can be incubated in a quenching buffer (e.g., 5% milk powder, 0.075% Tween20 in PBS) until the final mixture is added for incubation.

[0419] In our standard WB procedure, we use nitrocellulose membranes imprinted with HEK293T cell lysates. After pre-conjugation and purification or quenching of IgG: multivalent linker mixtures, they are pre-diluted in assay buffer (here: 5% BSA, 0.075% Tween20 in PBS solution), with a dilution twice the final dilution concentration (if the final concentration is 1: 1000, dilute to 1: 500 in this step). Then, the pre-diluted mixture is mixed with unconjugated competitive IgG (leakage experiment) or conjugated competitive IgG (multiple determination experiments) in 1: 1 to obtain the final dilution for WB incubation. Pre-diluted IgG: multivalent linker mixtures are mixed with a separate buffer in 1: 1 for use as a control.

[0420] The blots were incubated on a rotator at room temperature for 0.5-1.0 h. After incubation, the blots were washed three times with 0.075% Tween 20 in PBS for 5 minutes. The blots were dried using Whatman filter paper for at least 30 minutes before imaging.

[0421] Multiple membranes were blotted with different combinations of pre-labeled multivalent linker + primary antibody complexes.

[0422] Figure 4A and Figure 4C Depicted is the use of an anti-CoxIV primary antibody linked to a multivalent linker with a 488 fluorophore, as well as There were also membranes from the same species treated with a secondary anti-TDP primary antibody labeled with a multivalent linker with a 647 fluorophore. Figure 4B and Figure 4D The controls were treated with only the anti-CoxIV primary antibody linked to a multivalent linker with a 488 fluorophore or with the anti-TDP primary antibody labeled with a multivalent linker with a 647 fluorophore, respectively.

[0423] Figure 4A and Figure 4B shows the signal observed through the 488 excitation frequency channel, while Figure 4C and Figure 4DShown are the signals observed through the 647 excitation frequency channel. The results show that the membrane treated with two labeled primary antibodies from the same species did not exhibit any signal bleed-through.

[0424] Therefore, this example further demonstrates the use of the multivalent linkers of the present application in multiplex assays. Example 8: Multivalent Linkers in Fluorescence Activated Cell Sorting

[0425] Primary antibodies are often stored in glycerol to prevent antibody degradation; however, glycerol concentrations can inhibit various applications of primary antibodies. For example, glycerol can inhibit the function of reporter molecules (such as peroxidases), or inhibit recognition of target antigens. There are a variety of methods to remove glycerol from primary antigen solutions; however, the concentration of primary antibodies can be significantly reduced through these processes. Therefore, it is generally preferred to use reagents that are not negatively affected by glycerol. This example demonstrates that multivalent linkers are suitable for flow cytometry experiments at a wide range of different glycerol concentrations.

[0426] Standard flow cytometry procedures were performed. Briefly, cells were washed (single cell suspension) and cell numbers were adjusted to 1-5×10 in ice-cold FACS buffer (PBS, 0.5%-1% BSA or 5%-10% FBS, 0.1% NaN3 sodium azide). 6 The cells were stained in polystyrene round-bottom 12×75 mm BD Falcon tubes (Catalog No. 352052). Next, 100 μl of cell suspension was added to each tube. Next, 100 μl of Fc blocker (Fc blocker was diluted in FACS buffer at a ratio of 1:50) was added to each sample and incubated on ice for 20 min, followed by centrifugation at 1500 rpm for 5 min at 4°C.

[0427] To determine whether glycerol concentration affects the formation of a primary antibody-multivalent linker mixture, six different glycerol concentrations of an antigen solution were used: 0%, 5%, 10%, 15%, 20% and 25%. The multivalent linker was first conjugated to an allophycocyanin (APC) reporter. Then, the multivalent linker-APC reporter was incubated with a primary antibody targeting an intracellular epitope of interest to label the antibody. Next, 10 μg / ml of the primary antibody-multivalent linker complex was added to the cells and incubated in the dark for 30 min. The cells were washed 3 times by centrifugation at 1500 rpm for 5 minutes and resuspended in 1 ml of ice-cold FACS buffer. The cells were stored on ice in the dark until the scheduled analysis time.

[0428] like Fig. 6AAs shown in Table 3, different concentrations of glycerol (0-25%) in an antigen solution did not affect the number of positive cells or the ability to distinguish between labeled and unlabeled cell populations. Therefore, in FAC experiments, it is appropriate to form a primary antibody-multivalent linker mixture at a variety of glycerol concentrations.

[0429] Table 3. Glycerol concentrations of various monoantigen solutions in FAC experiments % (w / w) glycerol Q1％(+) 0 85.3 5 86.8 10 86.6 15 87.9 20 86.2 25 88.7

[0430] This experiment demonstrates that the primary antibody-multivalent linker mixture is suitable for flow cytometry using a wide range of primary antibody glycerol concentrations. Example 9: Other intended uses of multivalent linkers Immunohistochemistry

[0431] Multiplex assays using multivalent linkers can also be performed in immunocytochemistry experiments.

[0432] Tissues were fixed in paraformaldehyde (4% in buffer) using standard immunohistochemistry protocols and incubated at room temperature for approximately 2 hours.

[0433] Next, the paraformaldehyde was discarded and the tissue was washed 5 times in 0.1M phosphate buffer solution + 0.3% Triton-X for 10 minutes each time. The tissue was then embedded in paraffin. The paraffin-embedded tissue was sliced ​​using a microtome. Next, the paraffin-embedded tissue sections were transferred to a vial containing the first primary antibody-multivalent linker mixture and the second primary antibody-multivalent linker mixture and incubated overnight. After overnight incubation, the paraffin-embedded tissue was washed 6 times in Triton buffer for 10 minutes each time. The paraffin-embedded tissue sections were stored on ice or in the dark at 4°C until the scheduled analysis time.

[0434] Experiments will be performed using either a single linker-antibody combination or using multiple primary antibodies of the same species with multivalent linkers. No signal bleed-through or cross-reactivity is expected. Flow cytometry

[0435] Several flow cytometry examples will be performed to demonstrate the use of multivalent linkers in flow cytometry. In standard flow cytometry procedures, cells are washed (single cell suspension) and the cell number is adjusted to 1-5×10 in ice-cold FACS buffer (PBS, 0.5%-1% BSA or 5%-10% FBS, 0.1% NaN3 sodium azide). 6The cells were stained in polystyrene round-bottom 12×75 mm BD Falcon tubes (Catalog No. 352052). Next, 100 μl of cell suspension was added to each tube. Next, 100 μl of Fc blocker (Fc blocker was diluted in FACS buffer at a ratio of 1:50) was added to each sample and incubated on ice for 20 min, followed by centrifugation at 1500 rpm for 5 min at 4°C.

[0436] Next, add 0.1-10 μg / ml of the first primary antibody-multivalent linker mixture and 0.1-10 μg / ml of the second primary antibody-multivalent linker mixture and incubate in the dark for at least 30 min. If necessary, dilutions can be made in FACS buffer.

[0437] The cells were then washed three times by centrifugation at 1500 rpm for 5 minutes and resuspended in 200 μl to 1 ml of ice-cold FACS buffer. The cells were stored on ice or at 4°C in the dark until the scheduled analysis time.

[0438] Experiments will be performed using either a single linker-antibody combination or using multiple primary antibodies of the same species with multivalent linkers. No signal bleed-through or cross-reactivity is expected. Enzyme-linked immunosorbent assay (ELISA)

[0439] Enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the utility of the multivalent linker in this context.

[0440] In this ELISA study, the sample antigen, biotinylated GFP, was diluted in coating buffer at different concentrations. Each well of the plate was coated with 100 μL of coating solution. The plate was then covered and incubated at room temperature for one hour or overnight (12-18 hours) at 2-8°C.

[0441] The contents were then aspirated and the wells were washed once with >300 μL of wash buffer per well. After washing, the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0442] Subsequently, 300 μL of blocking buffer was added to each well for one hour at room temperature, after which the blocking buffer was aspirated and the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0443] Next, anti-GFP primary antibody plus HRP multivalent linker diluted in blocking buffer was added to each well and incubated for two hours at room temperature with gentle constant shaking (approximately 500 rpm).

[0444] The contents were then aspirated and washed five times with >300 μL of wash buffer per well. After washing, the plate was inverted and tapped on absorbent paper to remove excess liquid. The relative fluorescence units (RFU) were then measured on a standard plate reader. Results ( Figure 6B ) demonstrated excellent antigen binding measurements using primary antibodies labeled with multivalent linkers.

[0445] Another standard sandwich ELISA study will be performed to demonstrate the use of multivalent linkers in this context. In this study, the solution will be prepared by diluting the capture antibody in coating buffer. Each well of the plate will be coated with 100 μL of coating solution. The plate will then be covered and incubated at room temperature for one hour or overnight (12-18 hours) at 2-8°C.

[0446] The contents were then aspirated and the wells were washed once with >300 μL of wash buffer per well. After washing, the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0447] Subsequently, 300 μL of blocking buffer was added to each well for one hour at room temperature, after which the blocking buffer was aspirated and the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0448] Prepare standard and sample dilutions in blocking buffer. Pipette 100 μL of standards and samples (in duplicate) into designated wells. Incubate the plate at room temperature with gentle, continuous shaking (approximately 500 rpm) for one to two hours.

[0449] The contents were then aspirated and the wells were washed five times with >300 μL of wash buffer per well. After washing, the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0450] Next, the first primary antibody-multivalent linker mixture and the second primary antibody-multivalent linker mixture were diluted in blocking buffer, added to each well, and incubated at room temperature with gentle, continuous shaking (approximately 500 rpm) for two hours.

[0451] The contents were then aspirated and washed five times with >300 μL of wash buffer per well. After washing, the plate was inverted and tapped on absorbent paper to remove excess liquid.

[0452] For fluorescence-based reporters, relative fluorescence units (RFU) are measured on a standard plate reader.

[0453] Experiments will be performed using either a single linker-antibody combination or using multiple primary antibodies of the same species with multivalent linkers. No signal bleed-through or cross-reactivity is expected. Example 10: Joint section distance measurement

[0454] Understanding the exact epitope targeted by the multivalent linker helps calculate the linker segment length / distance factor in vivo. However, in the absence of such data, the epitope distance can be estimated by measuring the distance between equivalent positions on the target structure. Here, we use the crystal structure of complete mouse IgG1 (PDB ID 1igy; Harris et al. 1998, J.Mol.Biol.275:861-872) as a model to provide non-restrictive illustrative distance factors for the multivalent linker of the present disclosure. The focus is on the Fc fragment of IgG.

[0455] like Figure 5 As shown, the Fc fragment is an O-shaped homodimer with C2 symmetry, comprising the CH2 and CH3 domains of two γ heavy chains. The distance between the symmetry points varies significantly over the length of the Fc fragment, with the minimum (Euclidean) distance at the N-terminal and C-terminal tips being about 2-3 nm, but 5-7 nm in most other regions. Therefore, for most binding peptides, the linker segment may need to cover a Euclidean distance of at least 5 nm.

[0456] To provide additional flexibility to avoid steric hindrance (while taking into account epitope bridging of multivalent linkers), we designed linker segments with a length of about 5-9 nm based on Gly / Ser-rich sequences. We chose Gly and Ser because they confer the greatest flexibility (Gly) and hydrophilicity (Ser). With a peptide bond length of 0.38 nm, we designed linker segments with 15, 20, and 25 amino acids, which are equivalent to 5.7 nm, 7.6 nm, and 9.5 nm, respectively.

[0457] A multivalent linker with three linker segment lengths was generated. As shown in the experiment, a linker segment with 25 amino acids (9.5 nm) was within this calculation range and was tested in other parts of the specification (see, e.g. Figure 3 ).

[0458] As discussed above, a linker segment of insufficient size may result in aggregation of the primary antibody. Example 11: Biophysical Characterization of Multivalent Linkers

[0459] In the following examples, multivalent linkers were constructed based on camelid anti-antibody VHHs (also called single domain antibodies), which are variable domains of camelid heavy chain antibodies. VHHs that bind to IgG with very high affinity were selected to construct a bivalent form of a multivalent linker with a (G4S)5 linker segment: VHH1-(G4S)5-VHH2. This format is compatible with a 1:1 interaction of one IgG and one multivalent linker, because two multivalent linker VHH units can simultaneously occupy two equivalent epitopes on the IgG heteromer, for example on the two heavy chains of the Fc part. The multivalent linker also contains one or two cysteine ​​handles for reproducible site-specific maleimide conjugation of fluorophores or other labels. Two sets of multivalent linkers were designed, one set specific for rabbit IgG (which contains only one IgG isotype) and the other set specific for mouse IgG1 (which is the most common mouse IgG isotype).

[0460] The multivalent linker binds to the antibody with picomolar affinity and with a very slow off-rate. The binding kinetics of the multivalent linker were studied using biolayer interferometry (BLI) by titrating the multivalent linker onto the biotinylated binder immobilized on a streptavidin biosensor ( Figure 8 ). The observed dissociation rate k off Up to 10 -7 or 10 -6 s -1 , meaning that less than 5% of the multivalent linker dissociates from its antibody within hours to days. It is important to note that these off-rates were recorded at 30°C; like any other chemical rates, these rates will decrease further at lower temperatures (such as those typically used in multiplex immunostaining experimental settings). In addition, the on-rate of the multivalent linker is 10 5 Mol -1 s -1 The multivalent linker modifies its target primary antibody rapidly and, crucially, maintains binding to the same antibody over an extended period of time.

[0461] The 1:1 binding mode of the multivalent linker to IgG allows uniform labeling and maintains the oligomeric state of its target antibody. Dynamic light scattering (DLS) was used to analyze rabbit IgG in solution in both isolated state and in the state bound to the anti-rabbit IgG multivalent linker. Fig. 9As shown, the addition of the multivalent linker resulted in only a slight increase in the hydrodynamic radius Rh of this antibody, as expected for the formation of a 1:1 complex without aggregation or cross-linking. In contrast, when the rabbit IgG was mixed with a conventional secondary goat anti-rabbit IgG, the Rh increased significantly, indicating aggregation, which could result in loss of primary antibody activity in the presence of conventional secondary antibodies. Thus, the DLS results illustrate why conventional secondary antibodies must only be used in sequential staining protocols. In contrast, the binding mode of the multivalent linker is optimal for labeling antibodies prior to engagement with the target, allowing for multiplex immunostaining experiments. Example 12: Multiplex immunostaining using multivalent linkers

[0462] A rapid and reproducible multiplex immunostaining protocol was developed using high-affinity multivalent linkers against rabbit IgG and mouse IgG1. Fig.10 As summarized, the protocol consists of only two 5-min steps, namely the incubation of the multivalent linker with (1) its primary antibody and (2) a quencher. The quencher is either a complete IgG or an Fc fragment, acting as a decoy for the excess multivalent linker. In a first example, the protocol was applied to the simultaneous immunofluorescence co-staining of HeLa cells using: mouse monoclonal IgG1 primary antibodies against the mitochondrial protein HSP60 and the Golgi protein GORASP2, both of which were labeled with an anti-mouse IgG1 multivalent linker conjugated to 555 dye and an anti-mouse IgG1 conjugated to 647 dye, respectively. The resulting micrographs ( Fig.11A and Fig. 11B ) shows perfectly resolved mitochondria and Golgi cisternae. A two-dimensional cross-section of this image, the line intensity plot, shows a clear separation of the two signals ( Fig. 11C ), indicating that there was no cross-reaction between the two anti-mouse IgG1 multivalent linkers and the two mouse IgG1 primary antibodies.

[0463] In order to verify the labeling scheme, a quantitative and highly sensitive leakage assay was developed to check whether the multivalent linker bound to the first primary antibody would dissociate (leak) from the first primary antibody and then bind to the second primary antibody that was not labeled before. A systematic screening was carried out to test the general applicability of multivalent linkers in multiple immunostaining. Twenty-seven rabbit IgG and twenty-two mouse IgG1 primary antibodies were derived from a total of ten suppliers, covering monoclonal, polyclonal and recombinant antibodies produced and purified by different methods (see Table 4 below). It is worth noting that these primary antibodies were all stored according to the conditions provided by their suppliers without any further purification, buffer exchange or other treatment. Then, as described above, each primary antibody was successfully labeled with anti-rabbit IgG or anti-mouse IgG1 multivalent linkers, and the cross-reactivity of different primary antibodies with the same species / isotype was analyzed by using multiple assays with immunofluorescence. In all multiple assays, we observed staining of the expected target without any detectable cross-reaction, proving the superiority of primary antibody labeling based on multivalent linkers. Table 4. Summary of primary antibodies with multivalent linkers that were successfully tested in the leakage assay combined with immunofluorescence

[0464] This protocol provides a wide range of experimental flexibility. For example, the amount of primary antibody can be adjusted according to the needs of the assay. Commercial antibodies are provided in a very wide range of concentrations, provided that this information is given. Fig.12 As shown in Figure 2, primary antibodies provided at concentrations of 0.05 to 1 mg / ml can be labeled with multivalent linkers and successfully used to detect their target proteins. In addition, for some primary antibodies, adjusting the ratio of multivalent linkers to antibodies can further improve the overall intensity of the staining signal. In addition, the molecular complex formed by the primary antibody and the multivalent linker can be stable over an extended period of time ( Fig.13A ), as long as the primary antibody itself is sufficiently stable. Finally, limited access to suitable microscopy often delays the visualization of cell or tissue staining. Therefore, micrographs of immunofluorescence staining of multivalent linkers were recorded on the day of staining (day 0) and 42 days later, and no significant decrease in signal intensity was observed ( Fig. 13B ). In summary, this protocol can be tailored to the specific needs of an experiment, allowing multivalent linkers to be used in a wide range of applications. Example 13: Multivalent linker labeling of antibodies is compatible with a wide range of multiplex immunostaining applications

[0465] The multivalent linker labeling of antibodies was tested in a series of multiplex immunostaining applications. First, anti-mouse IgG1 or anti-rabbit IgG multivalent linkers were tested for immunofluorescence analysis of cells, where three ( Fig.14A ) or four ( Fig. 14B ) primary antibodies of the same species / isotype that visualize markers for the nucleolus, nuclear lamina, Golgi apparatus, and mitochondria. Primary antibodies of different species or isotypes can also be labeled with their respective polyvalent linkers, such as Fig.15A In addition, co-staining using multivalent linkers and chemically conjugated primary antibodies demonstrated that the antibody-multivalent linker complex is functionally equivalent to, and can be used together with, chemically labeled antibodies ( Fig. 15B ). In addition, multiplexed IHC / IF staining was successfully performed on various tissues, highlighting various cell types such as human renal tubules and glomeruli or rat brain neurons (Figure 16). It is noteworthy that the complex of the primary antibody with the multivalent linker is extremely stable, eliminating any cross-reactivity with endogenous IgG contained in the tissue. Therefore, prior Fc blocking is not necessary.

[0466] The above examples demonstrate multiple immunostaining of cultured cells or tissue sections using up to four different fluorophores introduced using our multivalent linkers. Multivalent linker biotin conjugates allow the experimenter to add additional fluorophores using streptavidin conjugates. In addition, cyclic IF (CyCIF, an overview is provided at the World Wide Web site: www.cycif.org) can also be used to further increase the level of multiplexing. In brief, CyCIF performs repeated staining cycles on samples, and the primary antibody used is labeled with a fluorophore or detected using a secondary antibody, followed by chemical bleaching (see, e.g., Lin et al., Elife. July 11, 2018; 7: e31657, the contents of which are incorporated by reference in their entirety). In a proof-of-principle experiment, the conjugated dye was shown to be effectively cleared using the CyCIF procedure, allowing multiple rounds of immunostaining ( Fig.17 ).

[0467] For super-resolution microscopy, primary antibodies labeled with dye-conjugated multivalent linkers will localize the fluorophore closer to its target because these primary antibodies labeled with dye-conjugated multivalent linkers are significantly smaller than the complex of the primary antibody and the full-length IgG secondary antibody, and therefore may lead to a significant improvement in image resolution. Example 14: Application of multivalent linkers in flow cytometry

[0468] Validation of multivalent linkers for use in flow cytometry. Flow cytometry and the related fluorescence activated cell sorting (FACS) benefit from the use of pre-labeled primary antibodies to minimize incubation times with live cells. In particular, multivalent linkers enable direct labeling of primary antibodies that are currently not available as chemical conjugates with suitable dyes.

[0469] In the first approach, it was confirmed in a modified leakage assay that the multivalent linker could label its target primary antibody without any cross-reactivity. Using the protocol described in the previous examples, a mouse IgG1 isotype control (non-binding monoclonal antibody) was labeled with an anti-mouse IgG1 multivalent linker conjugated to 650 dye, optionally treated with a quencher, and incubated with peripheral blood mononuclear cells (PBMCs) in the absence or presence of anti-CD3 mouse IgG1 monoclonal antibody. Multivalent linker labeling of anti-CD3 antibodies was effective in T cell subsets (i.e., CD3 + positive cells) produced a strong peak ( Fig.18 ), while in the presence of anti-CD3 antibody, the multivalent linker labeling of the isotype control had no signal, i.e., no leakage. Therefore, the multivalent linker system is compatible with flow cytometry.

[0470] Multivalent linkers enable multiplexed staining of cell surface receptors and intracellular markers in flow cytometry. Several scenarios for flow cytometry were tested with multivalent linkers. In the first study, three primary antibodies of different mouse isotypes (mouse IgG1, IgG2a, and IgG2b) were each labeled with the corresponding anti-mouse IgG1, IgG2a, or IgG2b multivalent linker labeled with 488, 555, or 647 dyes and used to stain PBMCs. Fig.19 As shown in Figure 2, this staining resulted in clear separation of CD45+ / CD3+CD3 / CD4+ (helper T cells) or CD3 / CD4- subsets. In a second study, four primary antibodies of the same isotype of mouse IgG1 were labeled with anti-mouse IgG1 multivalent linkers conjugated to FITC or 555, 647, or 750 dyes and used to stain PBMCs. Again, various populations such as helper T cells or cytotoxic T cells could be clearly identified ( Fig. 20 To quantify intracellular markers, rabbit polyclonal primary antibodies against human mitochondrial outer and inner membrane proteins were labeled with anti-rabbit IgG multivalent linkers conjugated to 647 dye and used to stain permeabilized HEK293T cells ( Fig.21 ). These examples demonstrate that labeling of primary antibodies with multivalent linkers provides comparable outcomes to directly conjugated antibodies in flow cytometry, but with the benefit of system flexibility.

[0471] The multivalent linker scheme was highly robust under a wide range of conditions. The compatibility of multivalent linkers with various additives that may be present in antibody formulations was investigated using the quantitative nature of flow cytometry. For example, anti-CD3 antibodies stored in 0%, 20%, or 50% glycerol were labeled using an anti-mouse IgG1 multivalent linker without removing the glycerol ( Fig. 22). No effect of glycerol was observed when T cell subsets of PBMCs were detected using a labeled UCTH1 antibody. Similarly, multivalent linker labeling of the anti-CD3 antibody OKT3 was not affected by high concentrations of BSA, up to 20%. Since primary antibodies can often be stored in the cell culture medium in which they were produced, we also tested the effects of three different cell culture media containing fetal bovine serum (FBS) on multivalent linker labeling of anti-CD3 antibodies. Again, multivalent linker labeling was highly effective in all conditions tested ( Fig. 22 ). Since additives such as glycerol, BSA, cell culture medium, and FBS are incompatible with traditional antibody labeling methods such as NHS conjugation, the high tolerance of the multivalent linker labeling protocol to these additives highlights its broad applicability.

[0472] In addition to excellent compatibility with various additives, factors that may affect the efficiency of cell staining for flow cytometry were also investigated in the multivalent linker labeling technology. Additives such as BSA, FBS, or EDTA can be included in flow cytometry or cell sorting experiments to maintain the cells to be analyzed. Therefore, mouse IgG1 anti-CD4 was labeled with an anti-mouse IgG1 multivalent linker conjugated to 647 dye to stain CD4 + PBMC staining. When the staining buffer was supplemented with increasing concentrations of BSA, FBS, or EDTA, or a combination thereof, the multivalent linker staining remained unaffected ( Fig.23 ). Example 15: Application of multivalent linkers in hybridoma screening

[0473] Multivalent linker labeling was found to be a robust technique for accelerating hybridoma cell screening. A critical and often time-consuming step in the process of creating new hybridoma cell lines is the screening of target-specific hybridoma cells. Chemical labeling of antibodies in hybridoma supernatants is almost impossible due to the composition of the culture medium. It is hypothesized that multivalent linker labeling circumvents this problem and allows direct labeling of antibodies in hybridoma supernatants. In the first approach, hybridoma supernatants were stimulated by titrating 0.0025-0.5μg of purified mouse IgG1 anti-CD3 primary antibody into 100μl of 1x RPMI cell culture medium supplemented with 15% FBS. Hybridoma supernatants were mock labeled for 5min using 1μl of anti-mouse IgG1 multivalent linker conjugated to 647 dye and used to stain PBMCs. The resulting flow cytometry signal increased proportionally to the amount of primary antibody ( Fig.24A). Notably, this reaction volume is an order of magnitude higher than the volume recommended by the standard protocol, again highlighting the flexibility of this system. Second, culture supernatants from hybridoma cells expressing mouse IgG2a anti-CD3 were diluted up to 128-fold in IMDM medium supplemented with 15% FBS and labeled using an anti-mouse IgG2a multivalent linker conjugated to 647 dye. All dilutions produced significant staining of PBMCs in flow cytometry ( Fig. 24B Similarly, supernatants from 100 to 10,000 hybridoma cells expressing mouse IgG1 anti-CD3 were labeled with multivalent linkers, and staining of PBMCs was successfully achieved for all cell numbers ( Fig.24C ). These data demonstrate that multivalent linkers allow direct labeling and detection of hybridoma supernatant antibodies over a wide range of cell numbers and expression levels. Thus, multivalent linkers enable rapid flow cytometric screening of hybridoma supernatants using a simple protocol that includes supplementing the supernatant (e.g., 100 μl) with 1 μl of multivalent linker, incubating at room temperature for 5 min, and using it to stain cells. Numbering Implementation Plan

[0474] Embodiment 1. A multivalent linker, wherein the multivalent linker specifically binds to a target antigen unit, wherein the multivalent linker comprises: a) a plurality of peptide binding arms, each binding arm being capable of binding to an epitope in the same target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

[0475] Embodiment 2. A molecular complex, comprising: (i) a single target antigen unit; and (ii) a multivalent linker, the multivalent linker comprising: a) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

[0476] Embodiment 3. The multivalent linker according to embodiment 1 or the molecular complex according to embodiment 2, wherein the multivalent linker has a molecular weight less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate to the target antigen unit, or wherein the multivalent linker is bound to the target antigen unit at a rate less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

[0477] Embodiment 4. The multivalent linker or molecular complex according to embodiment 3, wherein said k off (s-1) rates were determined by biolayer interferometry (BLI).

[0478] Embodiment 5. The multivalent linker according to any one of embodiments 1 and 3-4 or the molecular complex according to any one of embodiments 2-4, wherein the target antigen unit comprises a constant region of an antibody.

[0479] Embodiment 6. The multivalent linker of any one of embodiments 1 and 3-5 or the molecular complex of any one of embodiments 2-5, wherein the target antigen unit comprises the Fc region of an antibody.

[0480] Embodiment 7. A multivalent linker or molecular complex according to any one of Embodiments 5-6, wherein the epitope is located on the CH2 domain, CH3 domain and / or CH4 domain of the constant region or the Fc region.

[0481] Embodiment 8. A multivalent linker according to any one of embodiments 1 and 3-7 or a molecular complex according to any one of embodiments 2-7, wherein the one or more epitopes of the peptide binding arm are located on the CH1 domain or CL domain of the target antigen unit.

[0482] Embodiment 9. The multivalent linker of any one of embodiments 1 and 3-8 or the molecular complex of any one of embodiments 2-8, wherein the target antigen unit is an antibody, F(ab')2, Fab2, Fab3 or IgNAR.

[0483] Embodiment 10. The multivalent linker or molecular complex of Embodiment 9, wherein the antibody is IgG.

[0484] Embodiment 11. The multivalent linker or molecular complex according to embodiment 10, wherein the IgG antibody is (i) IgG1, IgG2, IgG3 or IgG4 subclass; optionally, wherein the IgG antibody is a human antibody; (ii) IgG1, IgG2a, IgG2b, IgG2c or IgG3 subclass; optionally, wherein the IgG antibody is a murine antibody; or (iii) IgG1, IgG2a, IgG2b or IgG2c subclass; optionally, wherein the IgG antibody is a rat antibody.

[0485] Embodiment 12. The multivalent linker or molecular complex of Embodiment 9, wherein the antibody is IgM.

[0486] Embodiment 13. The multivalent linker or molecular complex of any one of Embodiments 9-12, wherein the antibody is a heavy chain antibody.

[0487] Embodiment 14. A multivalent linker or molecular complex according to any one of Embodiments 9-13, wherein the antibody is a guinea pig antibody, a mouse antibody, a rat antibody, a chicken antibody (e.g., IgY), a donkey antibody, a rabbit antibody, a human antibody, a goat antibody, a pig antibody, a horse antibody, or a cow antibody.

[0488] Embodiment 15. A multivalent linker or molecular complex according to any one of Embodiments 9-14, wherein at least one of the peptide binding arms is cross-reactive and capable of non-simultaneously binding to antibodies from two or more species; optionally, wherein the two or more species are selected from humans, mice, rats and rabbits.

[0489] Embodiment 16. The multivalent linker or molecular complex of embodiment 15, wherein at least one of the peptide binding arms is cross-reactive and is capable of non-simultaneously binding antibodies from rabbit and human.

[0490] Embodiment 17. A multivalent linker according to any one of embodiments 1 and 3-16 or a molecular complex according to any one of embodiments 2-16, wherein each peptide binding arm is specific for a different epitope of the same target antigen unit.

[0491] Embodiment 18. A multivalent linker according to any one of embodiments 1 and 3-16 or a molecular complex according to any one of embodiments 2-16, wherein each peptide binding arm is specific for the same epitope, and wherein the target antigen unit comprises multiple identical epitopes.

[0492] Embodiment 19. A multivalent linker according to any one of embodiments 1 and 3-18 or a molecular complex according to any one of embodiments 2-18, wherein the multiple peptide binding arms are capable of binding to the same target antigen unit.

[0493] Embodiment 20. A multivalent linker according to any one of embodiments 1 and 3-19 or a molecular complex according to any one of embodiments 2-19, wherein the plurality of peptide binding arms do not bind to more than one target antigen unit.

[0494] Embodiment 21. The multivalent linker of any one of embodiments 1 and 3-20 or the molecular complex of any one of embodiments 2-20, wherein the multivalent linker is bivalent and comprises two peptide binding arms.

[0495] Embodiment 22. A multivalent linker according to any one of embodiments 1 and 3-21 or a molecular complex according to any one of embodiments 2-21, wherein less than 5%, 4%, 3%, 2% or 1% of the multiple peptide binding arms are cross-linked to different target antigen units.

[0496] Embodiment 23. A multivalent linker according to any one of embodiments 1 and 3-22 or a molecular complex according to any one of embodiments 2-22, wherein the multiple peptide binding arms are separated by a distance factor long enough to prevent the peptide binding arms from cross-linking with more than one target antigen unit.

[0497] Embodiment 24. The multivalent linker of any one of embodiments 1 and 3-23 or the molecular complex of any one of embodiments 2-23, wherein the linker segment comprises a peptide.

[0498] Embodiment 25. The multivalent linker according to any one of embodiments 1 and 3-24 or the molecular complex according to any one of embodiments 2-24, wherein the linker segment comprises between 5-50 amino acids.

[0499] Embodiment 26. The multivalent linker or molecular complex according to embodiment 25, wherein the linker segment comprises between 10-40 amino acids.

[0500] Embodiment 27. The multivalent linker or molecular complex according to embodiment 25, wherein the linker segment comprises between 20-30 amino acids.

[0501] Embodiment 28. The multivalent linker or molecular complex of any one of Embodiments 25-27, wherein the linker segment comprises about 25 amino acids, about 30 amino acids, or about 35 amino acids.

[0502] Embodiment 29. A multivalent linker according to any one of embodiments 1 and 3-28 or a molecular complex according to any one of embodiments 2-28, wherein in the extended conformation, the length of the linker segment is between.

[0503] Embodiment 30. A multivalent linker or molecular complex according to embodiment 29, wherein in the extended conformation, the length of the linker segment is about between.

[0504] Embodiment 31. A multivalent linker or molecular complex according to Embodiment 29, wherein in the extended conformation, the length of the linker segment is about between.

[0505] Embodiment 32. A multivalent linker or molecular complex according to Embodiment 29, wherein in the extended conformation, the length of the linker segment is about between.

[0506] Embodiment 33. A multivalent linker according to any one of embodiments 1 and 3-32 or a molecular complex according to any one of embodiments 2-32, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -5 , or wherein the multivalent linker is less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

[0507] Embodiment 34. A multivalent linker or molecular complex according to Embodiment 33, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -6 , or wherein the multivalent linker is less than or equal to 1.0×10 -6 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

[0508] Embodiment 35. The multivalent linker or molecular complex according to Embodiment 33, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -7 , or wherein the multivalent linker is less than or equal to 1.0×10 -7 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

[0509] Embodiment 36. A multivalent linker according to any one of embodiments 1 and 3-35 or a molecular complex according to any one of embodiments 2-35, wherein the linker segment comprises one (G4S) unit.

[0510] Embodiment 37. A multivalent linker or molecular complex according to Embodiment 36, wherein the linker segment comprises more than 2 (G4S) units.

[0511] Embodiment 38. A multivalent linker or molecular complex according to Embodiment 36, wherein the linker segment comprises more than 3 (G4S) units.

[0512] Embodiment 39. A multivalent linker or molecular complex according to Embodiment 36, wherein the linker segment comprises more than 4 (G4S) units.

[0513] Embodiment 40. A multivalent linker or molecular complex according to Embodiment 36, wherein the linker segment comprises more than 5 (G4S) units.

[0514] Embodiment 41. A multivalent linker or molecular complex according to Embodiment 36, wherein the linker segment comprises more than 6 (G4S) units.

[0515] Embodiment 42. A multivalent linker or molecular complex according to any one of Embodiments 36-41, wherein the linker segment comprises at most 4, at most 5, at most 6, at most 7, at most 8 or at most 9 (G4S) units.

[0516] Embodiment 43. A multivalent linker or molecular complex according to any one of embodiments 24-42, wherein the linker sequence comprises the amino acid sequence of GSTSGSGKSSEGKGEGSTSGSGKSG (SEQ ID NO: 495).

[0517] Embodiment 44. The multivalent linker or molecular complex according to any one of embodiments 24-43, wherein at least 20%-25% of the amino acids in the peptide of the linker segment are glycine.

[0518] Embodiment 45. The multivalent linker or molecular complex according to any one of embodiments 24-44, wherein between 60%-90% of the amino acids in the peptide of the linker segment are glycine.

[0519] Embodiment 46. A multivalent linker according to any one of embodiments 24-45, wherein between 10%-30% of the amino acids in the peptide of the linker segment are serine or threonine; more preferably, serine.

[0520] Embodiment 47. A multivalent linker according to any one of embodiments 24-46, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K) and arginine (R); more preferably, glycine, alanine, serine and threonine; more preferably, glycine, serine and threonine; more preferably, glycine and serine.

[0521] Embodiment 48. A multivalent linker according to any one of Embodiments 24-47, wherein the ratio of (i) glycine to (ii) serine and / or threonine in the linker segment is about 4:1.

[0522] Embodiment 49. The multivalent linker of any one of embodiments 1 and 3-48 or the molecular complex of any one of embodiments 2-48, wherein the multivalent linker comprises at least one moiety for conjugation to a heterologous molecule.

[0523] Embodiment 50. The multivalent linker or molecular complex of embodiment 49, wherein the moiety for conjugation is cysteine.

[0524] Embodiment 51. A multivalent linker or molecular complex according to embodiment 49, wherein the moiety for conjugation is lysine.

[0525] Embodiment 52. The multivalent linker or molecular complex of Embodiment 49, wherein the moiety for conjugation comprises biotin or streptavidin.

[0526] Embodiment 53. A multivalent linker or molecular complex according to embodiment 49, wherein the moiety for conjugation comprises a functional group for conjugation by click chemistry.

[0527] Embodiment 54. The multivalent linker or molecular complex of Embodiment 53, wherein the functional group comprises a dibenzocyclooctyne group (DBCO), an azide, a tetrazine and / or a trans-cyclooctene (TCO).

[0528] Embodiment 55. A multivalent linker or molecular complex according to any one of embodiments 49-54, wherein the linker segment or a peptide in the linker segment comprises the moiety for conjugation.

[0529] Embodiment 56. A multivalent linker or molecular complex according to any one of embodiments 49-55, wherein the heterologous molecule is a reporter, an oligonucleotide, a moiety functionalized for click chemistry, or an effector.

[0530] Embodiment 57. A multivalent linker according to any one of embodiments 1 and 3-56 or a molecular complex according to any one of embodiments 2-56, wherein the multivalent linker comprises at least one attached reporter, oligonucleotide, moiety functionalized for click chemistry, or effector.

[0531] Embodiment 58. A multivalent linker or molecular complex according to Embodiment 57, wherein the reporter is a fluorescent reporter.

[0532] Embodiment 59. The multivalent linker or molecular complex of Embodiment 58, wherein the fluorescent reporter is a fluorescein dye, a rhodamine dye, two or more fluorescent dyes that can act in concert with each other, or a protein that exhibits fluorescence.

[0533] Embodiment 60. A multivalent linker or molecular complex according to embodiment 58, wherein the fluorescent reporter is green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, mStrawberry, mTangerine and / or dsRed.

[0534] Embodiment 61. A multivalent linker or molecular complex according to Embodiment 57, wherein the reporter is an enzyme reporter.

[0535] Embodiment 62. A multivalent linker or molecular complex according to embodiment 61, wherein the enzyme reporter is horseradish peroxidase, cathepsin, matrix metalloproteinase, peptidase, carboxypeptidase, glycosidase, lipase, phospholipase, phosphatase, phosphodiesterase, sulfatase, reductase, bacterial enzyme, biotin ligase, DNA transposase or nuclease.

[0536] Embodiment 63. The multivalent linker or molecular complex of embodiment 62, wherein the DNA transposase is Tn5 transposase, or wherein the nuclease is micrococcal nuclease.

[0537] Embodiment 64. The multivalent linker or molecular complex of Embodiment 57, wherein the effector is a magnetic effector.

[0538] Embodiment 65. A multivalent linker or molecular complex according to embodiment 64, wherein the magnetic reporter is Gd(III), Dy(III), Fe(III), and Mn(II), DTPA, DOTA, DO3A, 2-benzyl-DOTA, α-(2-phenylethyl)1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA or 6,6″-bis[N,N,N″,N″-tetrakis(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

[0539] Embodiment 66. The multivalent linker of any one of embodiments 1 and 3-65 or the molecular complex of any one of embodiments 2-65, wherein the multivalent linker has an apparent K of the target antigen unit. DLess than 10,000 pM, less than 1,000 pM, less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 1 pM.

[0540] Embodiment 67. The multivalent linker or molecular complex of Embodiment 66, wherein the multivalent linker modifies the apparent K of the target antigen unit. D 1 to 10 pM, 10 to 50 pM, 50 to 100 pM, 100 to 500 pM or 500 to 1,000 pM.

[0541] Embodiment 68. The multivalent linker or molecular complex of Embodiment 66 or 67, wherein the multivalent linker modifies the apparent K of the target antigen unit. D Less than about 50 pM.

[0542] Embodiment 69. The multivalent linker or molecular complex of Embodiment 68, wherein the multivalent linker modifies the apparent K of the target antigen unit. D Less than about 25 pM.

[0543] Embodiment 70. The multivalent linker or molecular complex of Embodiment 69, wherein the multivalent linker modifies the apparent K of the target antigen unit. D Less than about 10 pM.

[0544] Embodiment 71. A multivalent linker according to any one of embodiments 1 and 3-70 or a molecular complex according to any one of embodiments 2-70, wherein the plurality of peptide binding arms comprises a peptide binding arm comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID No: 1-494.

[0545] Embodiment 72. A multivalent linker or molecular complex according to Embodiment 71, wherein the plurality of peptide binding arms comprises a peptide binding arm comprising a sequence selected from the group comprising SEQ ID No 1-494.

[0546] Embodiment 73. The multivalent linker of any one of embodiments 1 and 3-72 or the molecular complex of any one of embodiments 2-72, comprising more than one linker segment.

[0547] Embodiment 74. A multivalent linker according to any one of embodiments 1 and 3-73 or a molecular complex according to any one of embodiments 2-73, comprising the structure: (peptide binding arm)-linker segment-(peptide binding arm).

[0548] Embodiment 75. A multivalent linker according to any one of embodiments 1 and 3-73 or a molecular complex according to any one of embodiments 2-73, comprising the structure: (peptide binding arm)-linker segment-(peptide binding arm)-linker segment-(peptide binding arm).

[0549] Embodiment 76. The multivalent linker of any one of embodiments 1 and 3-75 or the molecular complex of any one of embodiments 2-75, wherein the peptide binding arm is a VHH of a camelid heavy chain antibody.

[0550] Embodiment 77. The multivalent linker of any one of embodiments 1 and 3-75 or the molecular complex of any one of embodiments 2-75, wherein the peptide binding arm is the VH of an immunoglobulin.

[0551] Embodiment 78. A multivalent linker according to any one of embodiments 1 and 3-77 or a molecular complex according to any one of embodiments 2-77, wherein the plurality of peptide binding arms are covalently attached to the linker segment.

[0552] Embodiment 79. A multivalent linker according to any one of embodiments 1 and 3-78 or a molecular complex according to any one of embodiments 2-78, wherein the plurality of peptide binding arms and the linker segment form a continuous polypeptide.

[0553] Embodiment 80. A multivalent linker according to any one of embodiments 1 and 3-79 or a molecular complex according to any one of embodiments 2-79, wherein the plurality of peptide binding arms and the linker segment are operably linked by translational fusion.

[0554] Embodiment 81. A multivalent linker or molecular complex according to any one of Embodiments 56-80, wherein the plurality of peptide binding arms, the linker segments, and the reporter or effector are each operably linked by translational fusion.

[0555] Embodiment 82. The multivalent linker of any one of embodiments 1 and 3-81 or the molecular complex of any one of embodiments 2-81, wherein each of the binding arms binds to an epitope of the target antigen unit.

[0556] Embodiment 83. A multivalent linker according to any one of embodiments 1 and 3-82 or a molecular complex according to any one of embodiments 2-82, wherein the peptide binding arm non-covalently binds to the epitope.

[0557] Embodiment 84. A composition comprising a multivalent linker according to any one of embodiments 1 and 3-83 or a molecular complex according to any one of embodiments 2-83.

[0558] Embodiment 85. A composition according to embodiment 84, comprising a buffer.

[0559] Embodiment 86. A composition comprising two or more different multivalent linkers according to any one of embodiments 1 and 3-83 or two or more different molecular complexes according to any one of embodiments 2-83, wherein each of the multivalent linkers is linked to a different reporter.

[0560] Embodiment 87. A composition according to any one of Embodiments 84-86, wherein the target antigen unit comprises a binding domain that is capable of binding to a test antigen after the multivalent linker is bound to the target antigen unit.

[0561] Embodiment 88. A composition according to Embodiment 87, further comprising a bait molecule, wherein the bait molecule comprises an epitope of the peptide binding arm or the plurality of peptide binding arms, but does not comprise the binding domain capable of binding to the test antigen.

[0562] Embodiment 89. The composition of any one of Embodiments 84-88, comprising a cryoprotectant selected from glycerol, ethylene glycol, and dimethyl sulfoxide (DMSO).

[0563] Embodiment 90. The composition of Embodiment 89, wherein the cryoprotectant is glycerol, and wherein the concentration of the glycerol is at most 50% by volume.

[0564] Embodiment 91. A composition according to embodiment 89, wherein the glycerol concentration is less than 30% or less than 15% by volume.

[0565] Embodiment 92. The composition of any one of embodiments 89-91, wherein the glycerol concentration is not less than 5% or not less than 10% by volume.

[0566] Embodiment 93. A method for detecting a test antigen in a sample, the method comprising the steps of: A) contacting the sample with a multivalent linker according to any one of embodiments 1 and 3-83 and a binding agent capable of specifically binding to the test antigen, and B) removing unbound binder and multivalent linker, and C) measuring the presence of the multivalent linker in the sample; wherein the binding agent comprises the target antigen unit, and wherein the binding agent specifically binds to the test antigen.

[0567] Embodiment 94. A method for detecting two or more test antigens in a sample, the method comprising contacting the sample with a first binding agent specific for a first test antigen and a second binding agent specific for a second test antigen, wherein the first binding agent and the second binding agent are each bound to a first multivalent linker and a second multivalent linker, respectively, wherein the first multivalent linker and / or the second multivalent linker is a multivalent linker according to any one of Embodiments 1 and 3-83, and wherein each multivalent linker is attached to a reporter, wherein the reporters are not the same.

[0568] Embodiment 95. The method according to embodiment 94, wherein each multivalent linker is less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate specifically binds to the constant region of the first binding agent or the second binding agent.

[0569] Embodiment 96. A method according to Embodiment 94 or 95, wherein the first binding agent and the second binding agent are each non-covalently bound to a first multivalent linker and a second multivalent linker, respectively.

[0570] Embodiment 97. A method according to Embodiment 94 or 95, wherein the first binding agent and the second binding agent are each linked or conjugated to a first multivalent linker and a second multivalent linker, respectively.

[0571] Embodiment 98. The method of any one of Embodiments 93-97, wherein less than 5%, 4%, 3%, 2% or 1% of the multivalent linkers are bound to two or more of the binding agents.

[0572] Embodiment 99. The method of any one of Embodiments 93-97, wherein less than 5%, 4%, 3%, 2% or 1% of the first multivalent linker is bound to the second binding agent, and wherein less than 5%, 4%, 3%, 2% or 1% of the second multivalent linker is bound to the first binding agent.

[0573] Embodiment 100. A method for detecting one or more test antigens in a sample, the method comprising contacting the sample with one or more molecular complexes according to any one of embodiments 2-83, wherein the single target antigen unit in each of the molecular complexes is contained in a binding agent, and wherein the binding agent is capable of specifically binding to the test antigen.

[0574] Embodiment 101. A method according to embodiment 100, which is used to detect two or more different test antigens using two or more of the molecular complexes, wherein the binding agent of each of the molecular complexes is capable of specifically binding to one of the test antigens.

[0575] Embodiment 102. A method according to any one of Embodiments 93-101, wherein the binding agent is an antibody or comprises an antigen-binding fragment thereof.

[0576] Embodiment 103. A method according to Embodiment 102, wherein the first antibody and the second antibody are antibodies of the same species.

[0577] Embodiment 104. A method according to Embodiment 102, wherein the first antibody and the second antibody are rabbit IgG antibodies.

[0578] Embodiment 105. A method according to Embodiment 102, wherein the first antibody and the second antibody are mouse IgG antibodies.

[0579] Embodiment 106. A method according to Embodiment 102, wherein the first antibody and the second antibody are rat IgG antibodies.

[0580] Embodiment 107. A method according to Embodiment 102, wherein the first antibody and the second antibody are human IgG antibodies.

[0581] Embodiment 108. The method of any one of Embodiments 94-107, wherein the first multivalent linker is incubated with the first binding agent prior to contacting the sample with the first binding agent.

[0582] Embodiment 109. A method according to Embodiment 108, wherein the second multivalent linker is incubated with the second binding agent before contacting the sample with the second binding agent.

[0583] Embodiment 110. The method of Embodiment 108 or 109, wherein the first binding agent is incubated with the first multivalent linker at a molar ratio of about 1:2.5.

[0584] Embodiment 111. The method of any one of Embodiments 108-110, wherein the second binding agent is incubated with the second multivalent linker at a molar ratio of about 1:2.5.

[0585] Embodiment 112. The method of any one of Embodiments 108-111, wherein the first binding agent has a stock solution concentration of at least 0.001 g / l.

[0586] Embodiment 113. The method of any one of Embodiments 108-112, wherein the second binding agent has a stock solution concentration of at least 0.001 g / l.

[0587] Embodiment 114. The method of any one of Embodiments 94-113, wherein the concentration of glycerol in the solution containing the first binding agent and / or the second binding agent is between 0-50% by volume.

[0588] Embodiment 115. The method of embodiment 114, wherein the glycerol concentration is less than 30% or less than 15% by volume.

[0589] Embodiment 116. The method of embodiment 114 or 115, wherein the glycerol concentration is not less than 5% or not less than 10% by volume.

[0590] Embodiment 117. A method according to any of Embodiments 93-116, wherein unbound multivalent linkers are quenched by adding a bait molecule that comprises an epitope of the peptide binding arm but does not bind to one or more of the test antigens.

[0591] Embodiment 118. The method of any one of Embodiments 93-117, wherein unbound multivalent linkers are removed from the multivalent linker-binder complex.

[0592] Embodiment 119. The method of Embodiment 118, wherein the unbound multivalent linker is removed by ultrafiltration.

[0593] Embodiment 120. A method according to embodiment 119, wherein the unbound multivalent linker is removed by bead depletion.

[0594] Embodiment 121. The method of any one of Embodiments 118-120, wherein the unbound multivalent linker is removed by adding non-specific polyclonal IgG or a fragment thereof.

[0595] Embodiment 122. The method of any one of Embodiments 118-120, wherein the unbound multivalent linker is removed by adding non-specific monoclonal IgG or a fragment thereof.

[0596] Embodiment 123. The method of any one of Embodiments 94-122, wherein the first multivalent linker and the first binding agent are incubated for about 30 minutes.

[0597] Embodiment 124. The method of any one of Embodiments 94-122, wherein the first multivalent linker and the first binding agent are incubated for less than 10 minutes.

[0598] Embodiment 125. The method of any one of Embodiments 94-124, wherein the second multivalent linker and the second binding agent are incubated for about 30 minutes.

[0599] Embodiment 126. The method of any one of Embodiments 94-124, wherein the second multivalent linker and the second binding agent are incubated for less than 10 minutes.

[0600] Embodiment 127. A method according to any one of embodiments 93-126, wherein the method is used for protein blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, immunohistochemistry, flow cytometry, fluorescence-assisted cell sorting (FACS), antibody screening (e.g., using hybridomas), spatial genomic analysis, or mass spectrometry.

[0601] Embodiment 128. A method according to embodiment 127, wherein the method is used for cyclic immunofluorescence detection.

[0602] Embodiment 129. A molecular complex comprising: (a) a single target antigen unit, the single target antigen unit comprising: i) a constant region or Fc region of an antibody, wherein the constant region or Fc region is selected from: 1) Human IgG1, IgG2, IgG3 or IgG4; 2) mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3; 3) Rat IgG1, IgG2a, IgG2b or IgG2c; or 4) rabbit IgG; and (b) a multivalent linker, the multivalent linker comprising: i) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit, wherein said peptide binding arm is a VHH of a camelid heavy chain antibody; and ii) at least one peptide linker segment operably covalently linked to the plurality of peptide binding arms, wherein the peptide linker segment is between 10 and 40 amino acids in length, and wherein at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K) and arginine (R).

[0603] Embodiment 130. A molecular complex according to Embodiment 129, wherein each of the peptide binding arms of the multivalent linker is non-covalently linked to the CH2 domain, CH3 domain and / or CH4 domain of the constant region or Fc region of the antibody.

[0604] Embodiment 131. A molecular complex according to any one of embodiments 129-130, wherein the linker segment comprises one (G4S) unit.

[0605] Embodiment 132. A molecular complex according to any one of embodiments 129-130, wherein the linker segment comprises more than 3 (G4S) units.

[0606] Embodiment 133. A molecular complex according to any one of embodiments 129-132, wherein the plurality of peptide binding arms and the peptide linker segment are operably linked by translational fusion. Incorporated by Reference

[0607] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, reference to any reference, article, publication, patent, patent publication and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1. A multivalent linker, wherein the multivalent linker specifically binds to a target antigen unit, and the multivalent linker comprises: a) a plurality of peptide binding arms, each binding arm being capable of binding to an epitope in the same target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

2. A molecular complex, comprising: (i) a single target antigen unit; and (ii) a multivalent linker, the multivalent linker comprising: a) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit; and b) at least one linker segment operably covalently linked to the plurality of peptide binding arms.

3. The multivalent linker according to claim 1 or the molecular complex according to claim 2, wherein the multivalent linker has a molecular weight less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate to the target antigen unit, or wherein the multivalent linker is bound to the target antigen unit at a rate less than or equal to 1.0×10 -4 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

4. The multivalent linker or molecular complex according to claim 3, wherein the k off (s -1 ) rates were determined by biolayer interferometry (BLI).

5. The multivalent linker according to any one of claims 1 and 3-4 or the molecular complex according to any one of claims 2-4, wherein the target antigen unit comprises a constant region of an antibody.

6. The multivalent linker according to any one of claims 1 and 3-5 or the molecular complex according to any one of claims 2-5, wherein the target antigen unit comprises the Fc region of an antibody.

7. The multivalent linker or molecular complex according to any one of claims 5 to 6, wherein the epitope is located on the CH2 domain, CH3 domain and / or CH4 domain of the constant region or the Fc region.

8. The multivalent linker according to any one of claims 1 and 3-7 or the molecular complex according to any one of claims 2-7, wherein the one or more epitopes of the peptide binding arm are located on the CH1 domain or CL domain of the target antigen unit.

9. The multivalent linker according to any one of claims 1 and 3-8 or the molecular complex according to any one of claims 2-8, wherein the target antigen unit is an antibody, F(ab')2, Fab2, Fab3 or IgNAR.

10. The multivalent linker or molecular complex of claim 9, wherein the antibody is IgG.

11. The multivalent linker or molecular complex according to claim 10, wherein the IgG antibody is (i) IgG1, IgG2, IgG3 or IgG4 subclass; optionally, wherein the IgG antibody is a human antibody; (ii) IgG1, IgG2a, IgG2b, IgG2c or IgG3 subclass; optionally, wherein the IgG antibody is a murine antibody; or (iii) IgG1, IgG2a, IgG2b or IgG2c subclass; optionally, wherein the IgG antibody is a rat antibody.

12. The multivalent linker or molecular complex of claim 9, wherein the antibody is IgM.

13. The multivalent linker or molecular complex according to any one of claims 9 to 12, wherein the antibody is a heavy chain antibody.

14. The multivalent linker or molecular complex according to any one of claims 9 to 13, wherein the antibody is a guinea pig antibody, a mouse antibody, a rat antibody, a chicken antibody (e.g., IgY), a donkey antibody, a rabbit antibody, a human antibody, a goat antibody, a pig antibody, a horse antibody, or a bovine antibody.

15. A multivalent linker or molecular complex according to any one of claims 9-14, wherein at least one of the peptide binding arms is cross-reactive and can non-simultaneously bind to antibodies from two or more species; optionally, wherein the two or more species are selected from humans, mice, rats and rabbits.

16. The multivalent linker or molecular complex according to claim 15, wherein at least one of the peptide binding arms is cross-reactive and is capable of non-simultaneously binding antibodies from rabbit and human.

17. The multivalent linker according to any one of claims 1 and 3-16 or the molecular complex according to any one of claims 2-16, wherein each peptide binding arm is specific for a different epitope of the same target antigen unit.

18. The multivalent linker according to any one of claims 1 and 3-16 or the molecular complex according to any one of claims 2-16, wherein each peptide binding arm is specific for the same epitope, and wherein the target antigen unit comprises a plurality of identical epitopes.

19. The multivalent linker according to any one of claims 1 and 3-18 or the molecular complex according to any one of claims 2-18, wherein the plurality of peptide binding arms are capable of binding to the same target antigen unit.

20. The multivalent linker according to any one of claims 1 and 3-19 or the molecular complex according to any one of claims 2-19, wherein the plurality of peptide binding arms do not bind to more than one target antigen unit.

21. The multivalent linker according to any one of claims 1 and 3-20 or the molecular complex according to any one of claims 2-20, wherein the multivalent linker is bivalent and comprises two peptide binding arms.

22. The multivalent linker of any one of claims 1 and 3-21 or the molecular complex of any one of claims 2-21, wherein less than 5%, 4%, 3%, 2% or 1% of the plurality of peptide binding arms are cross-linked to different target antigen units.

23. A multivalent linker according to any one of claims 1 and 3-22 or a molecular complex according to any one of claims 2-22, wherein the multiple peptide binding arms are separated by a distance factor long enough to prevent the peptide binding arms from cross-linking with more than one target antigen unit.

24. The multivalent linker of any one of claims 1 and 3-23 or the molecular complex of any one of claims 2-23, wherein the linker segment comprises a peptide.

25. The multivalent linker according to any one of claims 1 and 3-24 or the molecular complex according to any one of claims 2-24, wherein the linker segment comprises between 5-50 amino acids.

26. The multivalent linker or molecular complex of claim 25, wherein the linker segment comprises between 10-40 amino acids.

27. The multivalent linker or molecular complex of claim 25, wherein the linker segment comprises between 20-30 amino acids.

28. The multivalent linker or molecular complex of any one of claims 25-27, wherein the linker segment comprises about 25 amino acids, about 30 amino acids, or about 35 amino acids.

29. The multivalent linker according to any one of claims 1 and 3-28 or the molecular complex according to any one of claims 2-28, wherein in the extended conformation, the length of the linker segment is between.

30. The multivalent linker or molecular complex of claim 29, wherein in the extended conformation, the length of the linker segment is about between.

31. The multivalent linker or molecular complex of claim 29, wherein in the extended conformation, the length of the linker segment is about between.

32. The multivalent linker or molecular complex of claim 29, wherein in the extended conformation, the length of the linker segment is about between.

33. The multivalent linker according to any one of claims 1 and 3-32 or the molecular complex according to any one of claims 2-32, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -5 , or wherein the multivalent linker is less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

34. The multivalent linker or molecular complex of claim 33, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -6 , or wherein the multivalent linker is less than or equal to 1.0×10 -6 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

35. The multivalent linker or molecular complex of claim 33, wherein the apparent k of the multivalent linker is off (s -1 ) rate is less than or equal to 1.0×10 -7 , or wherein the multivalent linker is less than or equal to 1.0×10 -7 The apparent k off (s -1 ) rate dissociates from the single target antigen unit.

36. The multivalent linker according to any one of claims 1 and 3-35 or the molecular complex according to any one of claims 2-35, wherein the linker segment comprises one (G4S) unit.

37. The multivalent linker or molecular complex of claim 36, wherein the linker segment comprises more than 2 (G4S) units.

38. The multivalent linker or molecular complex of claim 36, wherein the linker segment comprises more than 3 (G4S) units.

39. The multivalent linker or molecular complex of claim 36, wherein the linker segment comprises more than 4 (G4S) units.

40. The multivalent linker or molecular complex of claim 36, wherein the linker segment comprises more than 5 (G4S) units.

41. The multivalent linker or molecular complex of claim 36, wherein the linker segment comprises more than 6 (G4S) units.

42. The multivalent linker or molecular complex of any one of claims 36-41, wherein the linker segment comprises at most 4, at most 5, at most 6, at most 7, at most 8 or at most 9 (G4S) units.

43. The multivalent linker or molecular complex of any one of claims 24-42, wherein the linker sequence comprises the amino acid sequence of GSTSGSGKSSEGKGEGSTSGSGKSG (SEQ ID NO: 495).

44. The multivalent linker or molecular complex according to any one of claims 24-43, wherein at least 20%-25% of the amino acids in the peptide of the linker segment are glycine.

45. The multivalent linker or molecular complex according to any one of claims 24-44, wherein between 60%-90% of the amino acids in the peptide of the linker segment are glycine.

46. ​​The multivalent linker of any one of claims 24-45, wherein between 10%-30% of the amino acids in the peptide of the linker segment are serine or threonine; more preferably, serine.

47. A multivalent linker according to any one of claims 24-46, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K) and arginine (R); more preferably, glycine, alanine, serine and threonine; more preferably, glycine, serine and threonine; more preferably, glycine and serine.

48. The multivalent linker of any one of claims 24-47, wherein the ratio of (i) glycine to (ii) serine and / or threonine in the linker segment is about 4:

1.

49. The multivalent linker of any one of claims 1 and 3-48 or the molecular complex of any one of claims 2-48, wherein the multivalent linker comprises at least one moiety for conjugation to a heterologous molecule.

50. The multivalent linker or molecular complex of claim 49, wherein the moiety for conjugation is cysteine.

51. The multivalent linker or molecular complex of claim 49, wherein the moiety for conjugation is lysine.

52. The multivalent linker or molecular complex of claim 49, wherein the moiety for conjugation comprises biotin or streptavidin.

53. The multivalent linker or molecular complex of claim 49, wherein the moiety for conjugation comprises a functional group for conjugation by click chemistry.

54. The multivalent linker or molecular complex of claim 53, wherein the functional group comprises a dibenzocyclooctyne group (DBCO), an azide, a tetrazine and / or a trans-cyclooctene (TCO).

55. The multivalent linker or molecular complex according to any one of claims 49-54, wherein the linker segment or a peptide in the linker segment comprises the moiety for conjugation.

56. The multivalent linker or molecular complex of any one of claims 49-55, wherein the heterologous molecule is a reporter, an oligonucleotide, a moiety functionalized for click chemistry, or an effector.

57. The multivalent linker of any one of claims 1 and 3-56 or the molecular complex of any one of claims 2-56, wherein the multivalent linker comprises at least one attached reporter, oligonucleotide, moiety functionalized for click chemistry, or effector.

58. The multivalent linker or molecular complex of claim 57, wherein the reporter is a fluorescent reporter.

59. The multivalent linker or molecular complex of claim 58, wherein the fluorescent reporter is a fluorescein dye, a rhodamine dye, two or more fluorescent dyes that can act in concert with each other, or a protein that exhibits fluorescence.

60. The multivalent linker or molecular complex of claim 58, wherein the fluorescent reporter is green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, mStrawberry, mTangerine and / or dsRed.

61. The multivalent linker or molecular complex of claim 57, wherein the reporter is an enzyme reporter.

62. The multivalent linker or molecular complex of claim 61, wherein the enzyme reporter is horseradish peroxidase, cathepsin, matrix metalloproteinase, peptidase, carboxypeptidase, glycosidase, lipase, phospholipase, phosphatase, phosphodiesterase, sulfatase, reductase, bacterial enzyme, biotin ligase, DNA transposase or nuclease.

63. The multivalent linker or molecular complex of claim 62, wherein the DNA transposase is Tn5 transposase, or wherein the nuclease is micrococcal nuclease.

64. The multivalent linker or molecular complex of claim 57, wherein the effector is a magnetic effector.

65. A multivalent linker or molecular complex according to claim 64, wherein the magnetic reporter is Gd(III), Dy(III), Fe(III), and Mn(II), DTPA, DOTA, DO3A, 2-benzyl-DOTA, α-(2-phenylethyl)1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tri(methylacetic acid), 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA or 6,6″-bis[N,N,N″,N″-tetrakis(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

66. The multivalent linker of any one of claims 1 and 3-65 or the molecular complex of any one of claims 2-65, wherein the multivalent linker has an apparent K of the target antigen unit. D Less than 10,000 pM, less than 1,000 pM, less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 1 pM.

67. The multivalent linker or molecular complex of claim 66, wherein the multivalent linker has an apparent K of the target antigen unit. D 1 to 10 pM, 10 to 50 pM, 50 to 100 pM, 100 to 500 pM or 500 to 1,000 pM.

68. The multivalent linker or molecular complex of claim 66 or 67, wherein the multivalent linker has an apparent K of the target antigen unit. D Less than about 50 pM.

69. The multivalent linker or molecular complex of claim 68, wherein the multivalent linker has an apparent K of the target antigen unit. D Less than about 25 pM.

70. The multivalent linker or molecular complex of claim 69, wherein the multivalent linker has an apparent K of the target antigen unit. D Less than about 10 pM.

71. A multivalent linker according to any one of claims 1 and 3-70 or a molecular complex according to any one of claims 2-70, wherein the plurality of peptide binding arms comprises a peptide binding arm comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID Nos: 1-494.

72. The multivalent linker or molecular complex of claim 71, wherein the plurality of peptide binding arms comprises a peptide binding arm comprising a sequence selected from the group comprising SEQ ID No 1-494.

73. The multivalent linker of any one of claims 1 and 3-72 or the molecular complex of any one of claims 2-72, comprising more than one linker segment.

74. The multivalent linker according to any one of claims 1 and 3-73 or the molecular complex according to any one of claims 2-73, comprising the structure: (peptide binding arm)-linker segment-(peptide binding arm).

75. The multivalent linker according to any one of claims 1 and 3-73 or the molecular complex according to any one of claims 2-73, comprising the structure: (peptide binding arm)-linker segment-(peptide binding arm)-linker segment-(peptide binding arm).

76. The multivalent linker according to any one of claims 1 and 3-75 or the molecular complex according to any one of claims 2-75, wherein the peptide binding arm is a VHH of a camelid heavy chain antibody.

77. The multivalent linker of any one of claims 1 and 3-75 or the molecular complex of any one of claims 2-75, wherein the peptide binding arm is a VH of an immunoglobulin.

78. The multivalent linker of any one of claims 1 and 3-77 or the molecular complex of any one of claims 2-77, wherein the plurality of peptide binding arms are covalently linked to the linker segment.

79. The multivalent linker of any one of claims 1 and 3-78 or the molecular complex of any one of claims 2-78, wherein the plurality of peptide binding arms and the linker segment form a continuous polypeptide.

80. The multivalent linker of any one of claims 1 and 3-79 or the molecular complex of any one of claims 2-79, wherein the plurality of peptide binding arms and the linker segment are operably linked by translational fusion.

81. The multivalent linker or molecular complex of any one of claims 56-80, wherein the plurality of peptide binding arms, the linker segments, and the reporter or effector are each operably linked by translational fusion.

82. The multivalent linker of any one of claims 1 and 3-81 or the molecular complex of any one of claims 2-81, wherein each of the binding arms binds to an epitope of the target antigen unit.

83. The multivalent linker of any one of claims 1 and 3-82 or the molecular complex of any one of claims 2-82, wherein the peptide binding arm non-covalently binds to the epitope.

84. A composition comprising the multivalent linker of any one of claims 1 and 3-83 or the molecular complex of any one of claims 2-83.

85. The composition of claim 84, comprising a buffer.

86. A composition comprising two or more different multivalent linkers according to any one of claims 1 and 3-83 or two or more different molecular complexes according to any one of claims 2-83, wherein each of the multivalent linkers is linked to a different reporter.

87. The composition of any one of claims 84-86, wherein the target antigen unit comprises a binding domain that is capable of binding to a test antigen after the multivalent linker is bound to the target antigen unit.

88. The composition of claim 87, further comprising a bait molecule comprising an epitope of the peptide binding arm or the plurality of peptide binding arms but not comprising the binding domain capable of binding to the test antigen.

89. The composition of any one of claims 84-88, comprising a cryoprotectant selected from glycerol, ethylene glycol, and dimethyl sulfoxide (DMSO).

90. The composition of claim 89, wherein the cryoprotectant is glycerol, and wherein the concentration of the glycerol is up to 50% by volume.

91. The composition of claim 89, wherein the glycerol concentration is less than 30% or less than 15% by volume.

92. The composition of any one of claims 89-91, wherein the glycerol concentration is not less than 5% or not less than 10% by volume.

93. A method for detecting a test antigen in a sample, the method comprising the steps of: A) contacting the sample with a multivalent linker according to any one of claims 1 and 3-83 and a binding agent capable of specifically binding to the test antigen, and B) removing unbound binder and multivalent linker, and C) measuring the presence of the multivalent linker in the sample; wherein the binding agent comprises the target antigen unit, and wherein the binding agent specifically binds to the test antigen.

94. A method for detecting two or more test antigens in a sample, the method comprising contacting the sample with a first binding agent specific for a first test antigen and a second binding agent specific for a second test antigen, wherein the first binding agent and the second binding agent are each bound to a first multivalent linker and a second multivalent linker, respectively, wherein the first multivalent linker and / or the second multivalent linker is a multivalent linker according to any one of claims 1 and 3-83, and wherein each multivalent linker is attached to a reporter, wherein the reporters are not the same.

95. The method of claim 94, wherein each multivalent linker is present in an amount less than or equal to 1.0×10 -5 The apparent k off (s -1 ) rate specifically binds to the constant region of the first binding agent or the second binding agent.

96. The method of claim 94 or 95, wherein the first binding agent and the second binding agent are each non-covalently bound to a first multivalent linker and a second multivalent linker, respectively.

97. The method of claim 94 or 95, wherein the first binding agent and the second binding agent are each linked or conjugated to a first multivalent linker and a second multivalent linker, respectively.

98. The method of any one of claims 93-97, wherein less than 5%, 4%, 3%, 2% or 1% of the multivalent linkers are bound to two or more of the binding agents.

99. The method of any one of claims 93-97, wherein less than 5%, 4%, 3%, 2% or 1% of the first multivalent linker is bound to the second binding agent, and wherein less than 5%, 4%, 3%, 2% or 1% of the second multivalent linker is bound to the first binding agent.

100. A method for detecting one or more test antigens in a sample, the method comprising contacting the sample with one or more molecular complexes according to any one of claims 2-83, wherein the single target antigen unit in each of the molecular complexes is contained in a binding agent, and wherein the binding agent is capable of specifically binding to the test antigen.

101. The method of claim 100, for detecting two or more different test antigens using two or more of said molecular complexes, wherein said binding agent of each of said molecular complexes is capable of specifically binding to one of said test antigens.

102. The method of any one of claims 93-101, wherein the binding agent is an antibody or comprises an antigen-binding fragment thereof.

103. The method of claim 102, wherein the first antibody and the second antibody are antibodies of the same species.

104. The method of claim 102, wherein the first antibody and the second antibody are rabbit IgG antibodies.

105. The method of claim 102, wherein the first antibody and the second antibody are mouse IgG antibodies.

106. The method of claim 102, wherein the first antibody and the second antibody are rat IgG antibodies.

107. The method of claim 102, wherein the first antibody and the second antibody are human IgG antibodies.

108. The method of any one of claims 94-107, wherein the first multivalent linker is incubated with the first binding agent prior to contacting the sample with the first binding agent.

109. The method of claim 108, wherein the second multivalent linker is incubated with the second binding agent prior to contacting the sample with the second binding agent.

110. The method of claim 108 or 109, wherein the first binding agent is incubated with the first multivalent linker at a molar ratio of about 1:2.

5.

111. The method of any one of claims 108-110, wherein the second binding agent is incubated with the second multivalent linker at a molar ratio of about 1:2.

5.

112. The method of any one of claims 108-111, wherein the first binding agent has a stock solution concentration of at least 0.001 g / l.

113. The method of any one of claims 108-112, wherein the second binding agent has a stock concentration of at least 0.001 g / l.

114. The method of any one of claims 94-113, wherein the concentration of glycerol in the solution containing the first binding agent and / or the second binding agent is between 0-50% by volume.

115. The method of claim 114, wherein the glycerol concentration is less than 30% or less than 15% by volume.

116. The method of claim 114 or 115, wherein the glycerol concentration is not less than 5% or not less than 10% by volume.

117. The method of any one of claims 93-116, wherein unbound multivalent linkers are quenched by adding a bait molecule that comprises an epitope of the peptide binding arm but does not bind to one or more of the test antigens.

118. The method of any one of claims 93-117, wherein unbound multivalent linkers are removed from the multivalent linker-binder complex.

119. The method of claim 118, wherein the unbound multivalent linker is removed by ultrafiltration.

120. The method of claim 119, wherein the unbound multivalent linker is removed by bead depletion.

121. The method of any one of claims 118-120, wherein the unbound multivalent linker is removed by adding non-specific polyclonal IgG or a fragment thereof.

122. The method of any one of claims 118-120, wherein the unbound multivalent linker is removed by adding non-specific monoclonal IgG or a fragment thereof.

123. The method of any one of claims 94-122, incubating the first multivalent linker and the first binding agent for about 30 minutes.

124. The method of any one of claims 94-122, wherein the first multivalent linker and the first binding agent are incubated for less than 10 minutes.

125. The method of any one of claims 94-124, wherein the second multivalent linker and the second binding agent are incubated for about 30 minutes.

126. The method of any one of claims 94-124, wherein the second multivalent linker and the second binding agent are incubated for less than 10 minutes.

127. The method of any one of claims 93-126, wherein the method is used for protein blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, immunohistochemistry, flow cytometry, fluorescence-assisted cell sorting (FACS), antibody screening (e.g., using hybridomas), spatial genomic analysis, or mass spectrometry.

128. The method of claim 127, wherein the method is used for cyclic immunofluorescence detection.

129. A molecular complex, comprising: (a) a single target antigen unit, the single target antigen unit comprising: i) a constant region or Fc region of an antibody, wherein the constant region or Fc region is selected from: 1) Human IgG1, IgG2, IgG3 or IgG4; 2) mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3; 3) Rat IgG1, IgG2a, IgG2b or IgG2c; or 4) rabbit IgG; and (b) a multivalent linker, the multivalent linker comprising: i) a plurality of peptide binding arms, wherein each peptide binding arm binds to said single target antigen unit, wherein said peptide binding arm is a VHH of a camelid heavy chain antibody; and ii) at least one peptide linker segment operably covalently linked to the plurality of peptide binding arms, wherein the peptide linker segment is between 10 and 40 amino acids in length, and wherein at least 80%, at least 85%, at least 90%, at least 95% or 100% of the amino acids in the linker segment are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), aspartic acid (D), lysine (K) and arginine (R).

130. The molecular complex of claim 129, wherein each of the peptide binding arms of the multivalent linker is non-covalently linked to a CH2 domain, a CH3 domain and / or a CH4 domain of a constant region or an Fc region of the antibody.

131. The molecular complex according to any one of claims 129-130, wherein the linker segment comprises one (G4S) unit.

132. The molecular complex according to any one of claims 129-130, wherein the linker segment comprises more than 3 (G4S) units.

133. The molecular complex of any one of claims 129-132, wherein the plurality of peptide binding arms and the peptide linker segment are operably linked by translational fusion.

Images