Method for purifying antibody-nucleic acid conjugates

Abstract

The present disclosure provides methods and kits relating to purification of antibody-nucleic acid conjugates using a PEG-based precipitation step.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 335,777, filed on April 28, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Antibody-nucleic acid conjugates can be used in a variety of situations, including but not limited to therapeutic applications of antisense oligonucleotides (ASOs) in which antibodies are used to specifically target specific cells (e.g., see Dugal-Tessier et al. J Clin Med. 2021 Feb 18; 10(4): 838), and diagnostic and research applications in which antibody-nucleic acid conjugates are used to detect target analytes in biological samples (e.g., see He et al. bioRxiv 2021 Nov 3, 467020, doi: 10.1101 / 2021.11.03.467020). Importantly, all of these uses require high-purity antibody-nucleic acid conjugates that are substantially free of contaminants (especially excess free nucleic acids). Therefore, there is a need in the art for improved, effective, and cost-effective methods and kits for purification of antibody-nucleic acid conjugates. The present disclosure addresses this need. Summary of the invention

[0004] The present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising an antibody-nucleic acid conjugate and an unconjugated nucleic acid, the method comprising: a) contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising an antibody-nucleic acid conjugate, an unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 5% and about 30% (w / v) and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution having a purity of at least 90% of the antibody-nucleic acid conjugate, as measured by size exclusion (SEC) high performance liquid chromatography (HPLC). In some aspects, the concentration of PEG in the mixture is between about 10% and about 25% (w / v). In some aspects, the concentration of NaCl in the mixture is between about 150 mM and about 200 mM.

[0005] In some aspects of the foregoing methods, the purified solution of the antibody-nucleic acid conjugate is at least 95% pure as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0006] In some aspects of the foregoing methods, step (b) comprises centrifuging the mixture at a speed of at least about 1,000 RCF (xg).

[0007] In some aspects of the foregoing methods, the centrifuging in step (b) is performed for at least about 2 minutes.

[0008] In some aspects of the foregoing methods, step (a) further comprises incubating the mixture at about 20°C for about 20 to about 40 minutes.

[0009] In some aspects of the foregoing methods, step (d) further comprises incubating the resuspended pellet at about 37°C for about 20 to about 40 minutes.

[0010] In some aspects of the foregoing methods, step (a) further comprises vortexing the mixture.

[0011] In some aspects of the foregoing methods, the resuspension solution comprises phosphate buffered saline (PBS).

[0012] In some aspects of the foregoing methods, the PEG is: i) PEG-6000; ii) PEG-8000; iii) PEG-10000; or iv) PEG-35,000. In some aspects, the PEG is PEG-6000. In some aspects, the PEG is PEG-8000.

[0013] In some aspects of the foregoing methods, contacting the solution with a purification solution in step (a) comprises contacting the solution with the purification solution at a ratio of 1:1 (v / v).

[0014] In some aspects of the foregoing methods, the concentration of PEG in the purification solution is between about 35% and about 45% (w / v). In some aspects of the foregoing methods, the concentration of PEG in the purification solution is about 40% (w / v).

[0015] In some aspects of the aforementioned methods, the concentration of NaCl in the purification solution is between about 150 mM and about 250 mM. In some aspects of the aforementioned methods, the concentration of NaCl in the purification solution is about 200 mM.

[0016] In some aspects of the aforementioned methods, the antibody comprises Fab, Fab', F(ab')2, Fv fragment, single-chain (sc)Fv (scFv) antibody fragment, linear antibody, single domain antibody (sdAb), Camelidae VHH domain, multispecific antibody, or any combination thereof.

[0017] In some aspects of the aforementioned methods, the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0018] In some aspects of the aforementioned methods, the nucleic acid comprises L-DNA. In some aspects of the aforementioned methods, the nucleic acid comprises D-DNA. In some aspects of the aforementioned methods, the nucleic acid comprises a combination of L-DNA and D-DNA.

[0019] In some aspects of the foregoing methods, the method is performed in one or more wells of a multi-well plate, thereby purifying multiple antibody-nucleic acid conjugates simultaneously.

[0020] In some aspects of the foregoing methods, the method is performed in two or more wells of a multi-well plate, thereby purifying multiple antibody-nucleic acid conjugates in different wells simultaneously.

[0021] The present disclosure provides a method for producing an antibody-nucleic acid conjugate, the method comprising: a) contacting a first solution comprising a nucleic acid comprising at least one reactive group with a second solution comprising an antibody under conditions such that at least one reactive group of the nucleic acid reacts with the antibody, thereby forming an antibody-nucleic acid conjugate; b) purifying the antibody-nucleic acid conjugate using any one of the purification methods described herein. In some aspects, at least one reactive group comprises a maleimide moiety.

[0022] The present disclosure provides a kit comprising: i) a first solution comprising a nucleic acid including at least one reactive group; and ii) a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl).

[0023] In some aspects of the foregoing kits, the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0024] In some aspects of the aforementioned kits, the nucleic acid comprises L-DNA. In some aspects of the aforementioned kits, the nucleic acid comprises D-DNA. In some aspects of the aforementioned kits, the nucleic acid comprises a combination of L-DNA and D-DNA.

[0025] In some aspects of the foregoing kits, at least one reactive group comprises a maleimide moiety.

[0026] In some aspects, the aforementioned kit further comprises a second solution comprising an antibody.

[0027] In some aspects of the aforementioned kits, the antibody comprises Fab, Fab', F(ab')2, Fv fragment, single-chain (sc)Fv (scFv) antibody fragment, linear antibody, single domain antibody (sdAb), Camelidae VHH domain, multispecific antibody, or any combination thereof.

[0028] In some aspects, the aforementioned kit further comprises a third solution comprising a nucleic acid comprising at least one reactive group, wherein the nucleic acid in the first solution comprises a nucleic acid sequence different from the nucleic acid sequence of the nucleic acid in the third solution.

[0029] In some aspects of the aforementioned kits, the PEG is: i) PEG-6000; ii) PEG-8000; iii) PEG-10000; or iv) PEG-35,000. In some aspects, the PEG is PEG-6000. In some aspects, the PEG is PEG-8000.

[0030] In some aspects of the aforementioned kits, the concentration of PEG in the purification solution is between about 35% to about 50% (w / ). In some aspects of the aforementioned kits, the concentration of PEG in the purification solution is about 40% (w / v).

[0031] In some aspects of the aforementioned kits, the concentration of NaCl in the purification solution is between about 150 mM-250 mM. In some aspects of the aforementioned kits, the concentration of NaCl in the purification solution is about 200 mM.

[0032] In some aspects, the aforementioned kits further comprise at least one aliquot of TCEP.

[0033] In some aspects, the aforementioned kits further comprise at least one spin filter.

[0034] In some aspects, the aforementioned kits further comprise at least one multi-well plate.

[0035] Any aspect and / or embodiment described above and herein may be combined with any other aspect and / or embodiment described above and herein.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. In this specification, the singular also includes the plural, unless the context clearly specifies otherwise; for example, the terms "a", "an" and "the" are understood to be singular or plural and the term "or" is understood to be included. For example, "an element" means one or more elements. Throughout this specification, the word "comprising" or variants such as "comprises" or "comprising" will be understood to mean including the elements, integers or steps or groups of elements, integers or steps, but not excluding any other elements, integers or steps or groups of elements, integers or steps. Approximately can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value. Throughout this specification, statements describing a range of values ​​as "between x and y" or "between x to y," where x and y are two values, are understood to include x and y. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0037] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are combined in their entirety by reference for all purposes. It is not recognized that the references cited herein are prior art for the claimed invention. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and examples are only illustrative and intended to be restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] When together with the attached Figure 1 The above and further features will be more clearly appreciated from the following detailed description.

[0040] Figure 1 SEC HPLC chromatogram of unconjugated antibody is shown.

[0041] Figure 2 SEC HPLC chromatogram of the unpurified antibody / nucleic acid conjugation reaction is shown.

[0042] Figure 3 SEC HPLC chromatogram showing an antibody / nucleic acid conjugate reaction purified using the methods of the present disclosure, wherein no NaCl was used during purification, resulting in little or no purification of the antibody-nucleic acid conjugate.

[0043] Figure 4 SEC HPLC chromatogram showing an antibody / nucleic acid conjugate reaction purified using the disclosed method, wherein NaCl was used during purification, resulting in purification of the antibody-nucleic acid conjugate to purity levels exceeding 90%.

[0044] Figure 5 Shown are SEC HPLC chromatograms of two antibody / nucleic acid conjugate reactions purified using the disclosed methods and an unpurified antibody-nucleic acid conjugate reaction.

[0045] Figure 6 Graph comparing the activity of antibody-nucleic acid conjugates purified using the disclosed method (y-axis) or existing SEC HPLC method (x-axis).

[0046] Fig. 7A Graph comparing the activity of antibody-nucleic acid conjugates purified using the disclosed methods in separate forms (y-axis) and in combined forms (x-axis) in NK92 cells.

[0047] Figure 7B Graph comparing the activity of antibody-nucleic acid conjugates purified using the disclosed methods in individual forms (y-axis) and in combined forms (x-axis) in HCC78 cells. DETAILED DESCRIPTION

[0048] The present disclosure is based, at least in part, on methods and kits for purifying antibody-nucleic acid conjugates.

[0049] As the skilled person will appreciate, existing methods for producing and subsequently purifying antibody-nucleic acid conjugates have various shortcomings that limit their availability. For example, many existing methods for purifying antibody-nucleic acid conjugates rely on the use of size exclusion chromatography HPLC, that is, based on size, antibody-nucleic acid conjugates are separated from free nucleic acids and / or free antibodies. However, SECHPLC requires expensive equipment, requires specialized training and is not easy to expand (each sample must be processed separately by SEC HPLC columns). Other existing methods for purifying antibody-nucleic acid conjugates use affinity chromatography. However, the affinity matrix used in these methods may be expensive and often requires the use of harsh elution conditions, which may damage the antibody-nucleic acid conjugates.

[0050] In contrast to these existing methods, the present disclosure provides antibody-nucleic acid conjugate purification methods based on polyethylene glycol (PEG) precipitation and related kits and compositions. Compared with the described existing purification methods, the antibody-nucleic acid conjugate purification methods presented herein are cheaper, faster and easier to implement, while still providing a high purity level. In addition, the method described herein does not rely on the use of harsh conditions, thereby preventing damage to the antibody-nucleic acid conjugate. Finally, unlike the existing methods, the purification method described herein is scalable, and can even be carried out in microplates (e.g., 6, 12, 24, 48, 96, 384, 1536, 3456 and 9600 well plates). Through scalability, the purification method described herein can be used to simultaneously label various antibodies. This application is particularly useful in diagnosis and research situations, where users may expect to use tens to hundreds of different antibody-nucleic acid conjugates to understand (interrogate) tens to hundreds of different molecular targets.

[0051] Purification method disclosed herein

[0052] The present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution containing the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution containing polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture containing the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution.

[0053] The present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution containing the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution containing polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture containing the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purification solution.

[0054] As described above, unlike existing methods known in the art, the purification methods disclosed herein are easily scalable, allowing for simultaneous or nearly simultaneous labeling of multiple different antibody species. To this end, any purification method described herein can be performed in at least one well of a microwell plate, preferably simultaneously or nearly simultaneously in multiple wells of a microwell plate. The purification methods described herein can be performed in a multi-tube array, a multi-tube array, or a similar structure. For ease of description, the terms "microwell plate" or "multi-well plate" or "multi-tube array" are used substantially interchangeably herein. The precise structure used will depend on the embodiment.

[0055] For example, the microplate can be a 6-well plate, a 12-well plate, a 48-well plate, a 96-well plate, a 384-well plate, a 1536-well plate, a 3456-well plate, or a 9600-well plate.

[0056] Therefore, the present disclosure provides a method for purifying multiple antibody-nucleic acid conjugates, the method comprising: a) contacting a solution including an antibody-nucleic acid conjugate and an unconjugated nucleic acid with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture including an antibody-nucleic acid conjugate, an unconjugated nucleic acid, PEG and NaCl; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution, wherein steps (a)-(e) are performed in multiple wells to produce multiple purified solutions. In some aspects of the aforementioned method, any combination of steps (a)-(e) can be performed simultaneously or almost simultaneously in multiple wells of a multi-well plate. In some aspects of the aforementioned method, some of steps (a)-(e) can be performed sequentially in multiple wells in a multi-well plate, while the remaining steps are performed simultaneously or almost simultaneously.

[0057] Therefore, the present disclosure provides a method for purifying multiple antibody-nucleic acid conjugates, the method comprising: a) contacting a solution including antibody-nucleic acid conjugates and unconjugated nucleic acids with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture including antibody-nucleic acid conjugates, unconjugated nucleic acids, PEG and NaCl; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; wherein steps (a)-(d) are performed in multiple wells to produce multiple purified solutions. In some aspects of the foregoing method, any combination of steps (a)-(d) can be performed simultaneously or almost simultaneously in multiple wells of a multi-well plate. In some aspects of the foregoing method, some of steps (a)-(d) can be performed sequentially in multiple wells of a multi-well plate, while the remaining steps are performed simultaneously or almost simultaneously.

[0058] In some aspects of the aforementioned method, the antibodies of the antibody-nucleic acid conjugates located in different holes may be different. In a non-limiting example, the first hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a first antibody bound to a first protein target, and the second hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a second antibody bound to a second protein target, wherein the first protein target and the second protein target are different. In some aspects, a plurality of holes in a multiwell plate may contain a solution comprising an antibody-nucleic acid conjugate with the same antibody, and other holes in the multiwell plate may contain a solution comprising an antibody-nucleic acid conjugate containing different antibodies. In a non-limiting example, the first hole of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a first antibody bound to a first protein target, the second hole of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a first antibody bound to a first protein target, and the third hole of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a second antibody bound to a second protein target, wherein the first protein target and the second protein target are different. In another non-limiting example, a first well of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a first antibody that binds to a first protein target, a second well of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a second antibody that binds to a second protein target, and a third well of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a third antibody that binds to a third protein target, wherein the first protein target, the second protein target, and the third protein target are different. The skilled artisan will appreciate that in this manner, the purification methods described herein allow for rapid and simultaneous purification of multiple antibody-nucleic acid conjugates, wherein the multiple conjugates comprise multiple different conjugate species that bind to multiple different molecular targets. This application is particularly useful in the diagnostic and research uses described above, where users often have knowledge of the presence, absence, and / or location of multiple different molecular targets in a biological sample.

[0059] In some aspects, multiple antibody-nucleic acid labeling reactions can be pooled together prior to performing any of the purification methods disclosed herein, such that multiple different antibody-nucleic acid conjugates are purified simultaneously in the same solution using the methods disclosed herein.

[0060] In a similar manner, in some aspects of the aforementioned method, the nucleic acid of the antibody-nucleic acid conjugate located in different holes of the multiwell plate may be different and / or distinguishable in at least a portion of its sequence. In a non-limiting example, the first hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid including a first nucleic acid sequence, and the second hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid including a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are different. In some aspects, a plurality of holes in the multiwell plate may contain a solution comprising an antibody-nucleic acid conjugate having a nucleic acid including a first nucleic acid sequence, and other holes in the multiwell plate may contain a solution comprising an antibody-nucleic acid conjugate having a nucleic acid including a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are different. In a non-limiting example, the first hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid including a first nucleic acid sequence, the second hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid including a first nucleic acid sequence, and the third hole of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid including a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are different. In another non-limiting example, a first well of a multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid comprising a first nucleic acid sequence and a second nucleic acid sequence, a second well of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid comprising the first nucleic acid sequence and the second nucleic acid sequence, and a third well of the multiwell plate may contain an antibody-nucleic acid conjugate comprising a nucleic acid comprising the second nucleic acid sequence and a third nucleic acid sequence, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are different.

[0061] In some aspects of the foregoing methods, step (a) comprises a solution and a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 5% and about 30% (w / v), or such that the concentration of PEG in the mixture is between about 7.5% and about 27.5% (w / v), or between about 10% and about 25% (w / v), or between about 12.5% ​​and about 22.5% (w / v), or between about 15% and about 20% (w / v), or between about 5% and about 25% (w / v). In some aspects, the concentration of PEG in the mixture can be any range within the above-described range. In some aspects, the concentration of PEG in the mixture is about 10% to about 25% (w / v).

[0062] In some aspects of the foregoing methods, step (a) comprises contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG, and NaCl, such that the concentration of NaCl in the mixture is between about 150 mM and about 400 mM, or between about 175 mM and about 375 mM, or between about 200 mM and about 350 mM, or between about 225 mM and about 325 mM, or between about 250 mM and about 300 mM, or between about 150 mM and about 350 mM, or between about 175 mM and about 350 mM, or between about 225 mM and about 350 mM, or between about 250 mM and about 350 mM, or between about 275 mM and about 350 mM, or between about 300 mM and about 350 mM, or between about 325 mM and about 350 mM. Occasionally between about 200mM to about 325mM or between about 200mM to about 300mM or between about 200mM to about 275mM or between about 200mM to about 250mM or between about 200mM to about 250mM or between about 200mM to about 225mM or between about 100mM to about 250mM or between about 125mM to about 225mM or between about 150mM to about 200mM or In some aspects, the concentration of NaCl in the mixture can be any range within the above description range. In some aspects, the concentration of NaCl in the mixture is about 200mM-about 350mM. In some aspects, the concentration of NaCl in the mixture is about 100mM-about 250mM.

[0063] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10% and about 25% and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution.

[0064] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10% and about 25% and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purification solution.

[0065] In some aspects, the purified solution may include a concentration of about 30% or about 31% or about 32% or about 33% or about 34% or about 35% or about 36% or about 37% or about 38% or about 39% or about 40% or about 41% or about 42% or about 43% or about 44% or about 45% or about 46% or about 47% or about 48% or about 49% or about 50% (w / v) PEG. In some aspects, the purified solution may include a concentration of about 30%-about 50% or about 35%-about 45% PEG. In some aspects, the purified solution may include a concentration of about 40% PEG.

[0066] In some aspects, the purified solution may include a concentration of about 150mM or about 155mM or about 160mM or about 165mM or about 170mM or about 175mM or about 180mM or about 185mM or about 190mM or about 195mM or about 200mM or about 205mM or about 210mM ​​or about 215mM or about 220mM or about 225mM or about 230mM or about 235mM or about 240mM or about 245mM or about 250mM NaCl. In some aspects, the purified solution may include a concentration of about 150mM-about 250mM or about 175mM-about 225mM NaCl. In some aspects, the purified solution may include a concentration of about 200mM NaCl.

[0067] Thus, in some aspects, the purification solution may comprise a PEG concentration between about 30% and about 50% (w / v) and a NaCl concentration between about 150 mM and about 250 mM. In some aspects, the purification solution may comprise a PEG concentration of about 40% (w / v) and a NaCl concentration of about 200 mM.

[0068] In some aspects of the foregoing methods, step (b) may comprise centrifuging the mixture at a speed of at least about 1,000 RCF (xg) or at least about 5,000 RCF (xg) or at least about 10,000 RCF (xg) or at least about 50,000 RCF (xg) or at least about 100,000 RCF (xg).

[0069] In some aspects of the foregoing methods, step b) may comprise centrifuging the mixture for at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 8.5 minutes, at least about 9 minutes, at least about 9.5 minutes, or at least about 10 minutes.

[0070] Thus, in some aspects of the foregoing methods, step (b) can include centrifuging the mixture at a speed of at least about 1,000 RCF (xg) for at least about 2 minutes.

[0071] In some aspects of the foregoing methods, step b) may comprise centrifuging the mixture for about 30 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 8 minutes, about 8.5 minutes, about 9 minutes, about 9.5 minutes, or about 10 minutes.

[0072] Thus, in some aspects of the foregoing methods, step (b) can include centrifuging the mixture at a speed of at least about 1,000 RCF (xg) for about 2 minutes.

[0073] As will be appreciated by the skilled artisan, centrifuging the mixture may include using centrifugation methods and centrifugation devices known in the art, including but not limited to low speed centrifugation, high speed centrifugation, ultracentrifugation, fixed angle rotor centrifugation, swinging bucket rotor centrifugation, swing-out rotor centrifugation, vertical rotor centrifugation, benchtop centrifuge, continuous flow centrifuge, gas centrifuge, microcentrifuge, refrigerated centrifuge, vacuum centrifuge, or any combination thereof.

[0074] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution containing the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution containing polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture containing the antibody-nucleic acid conjugate, the unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10%-about 25% and the concentration of NaCl in the mixture is between about 200mM-350mM; b) centrifuging the mixture at a speed of at least about 1,000RCF to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purification solution of step (d) to produce a purified solution.

[0075] As will be appreciated by those skilled in the art, polyethylene glycol (PEG) is a polymer of ethylene oxide. PEG can be obtained with a variety of different molecular weights based on the length of the polymer. Therefore, in some aspects of the aforementioned method, PEG can be PEG-100, PEG-200, PEG-300, PEG-400, PEG-500, PEG-600, PEG-700, PEG-800, PEG-900, PEG-1000, PEG-1500, PEG-2000, PEG-3000, PEG-3350, PEG-4000, PEG-5000, PEG-6000, PEG-7000, PEG-8000, PEG-9000, PEG-10000, PEG-15000, PEG-20000 or PEG-35000. In some aspects, PEG can be PEG-6000. In some aspects, PEG can be PEG-8000.

[0076] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution comprising polyethylene glycol (PEG)-6000 and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, the unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10%-about 25% and the concentration of NaCl in the mixture is between about 200mM-350mM; b) centrifuging the mixture at a speed of at least about 1,000RCF to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution.

[0077] Accordingly, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution comprising polyethylene glycol (PEG)-6000 and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10% and about 25% and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM; b) centrifuging the mixture at a speed of at least about 1,000 RCF to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspension solution to produce a purification solution.

[0078] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution comprising polyethylene glycol (PEG)-8000 and sodium chloride (NaCl) to produce a mixture comprising the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10%-about 25% and the concentration of NaCl in the mixture is between about 200mM-350mM; b) centrifuging the mixture at a speed of at least about 1,000RCF to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purification solution of step (d) to produce a purified solution.

[0079] Therefore, the present disclosure provides a method for purifying an antibody-nucleic acid conjugate from a solution containing the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising a) contacting the solution with a purification solution containing polyethylene glycol (PEG)-8000 and sodium chloride (NaCl) to produce a mixture containing the antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 10%-about 25% and the concentration of NaCl in the mixture is between about 100 mM-about 250 mM; b) centrifuging the mixture at a speed of at least about 1,000 RCF to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspension solution to produce a purification solution.

[0080] In some aspects of the foregoing methods, contacting the solution with a purification solution in step (a) may comprise contacting the solution with the purification solution at a ratio of about 0.1:1 (v / v), or about 0.5:1 (v / v), or about 1:1 (v / v), or about 1.5:1 (v / v), or about 2:1 (v / v), or about 3:1 (v / v), or about 4:1 (v / v), or about 5:1 (v / v), or about 10:1 (v / v), or about 1:0.1 (v / v), or about 1:0.5 (v), or about 1:1.5 (v / v), or about 1:2 (v / v), or about 1:3 (v / v), or about 1:4 (v / v), or about 1:5 (v / v), or about 1:10 (v / v). In some aspects of the foregoing methods, contacting the solution with a purification solution in step (a) can include contacting the solution with the purification solution at a ratio of about 1:1 (v / v).

[0081] In some aspects of the aforementioned methods, the solution comprising an antibody-nucleic acid conjugate and unconjugated nucleic acid may further comprise unconjugated antibody. In some aspects of the aforementioned methods, the mixture comprising an antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl may further comprise unconjugated antibody.

[0082] In some aspects of the aforementioned method, step (a) may further include vortexing and / or stirring the mixture after the solution contacts the purified solution. Without wishing to be bound by theory, by vortexing and / or stirring the mixture after the solution contacts the purified solution, it is ensured that the mixture is fully homogenized. Vortexing and / or stirring can be completed using any method known in the art, and can be carried out for at least about 1 second or at least about 2 seconds or at least about 3 seconds or at least about 4 seconds or at least about 5 seconds or at least about 6 seconds or at least about 7 seconds or at least about 8 seconds or at least about 9 seconds or at least about 10 seconds or about 1 second or about 2 seconds or about 3 seconds or about 4 seconds or about 5 seconds or about 6 seconds or about 7 seconds or about 8 seconds or about 9 seconds or about 10 seconds.

[0083] In some aspects of the foregoing methods, step (a) may comprise incubating the mixture for between about 10 minutes and about 50 minutes, or between about 15 minutes and about 45 minutes, or between about 20 and about 40 minutes, or between about 25 and about 35 minutes, or between about 20 minutes and about 50 minutes, or between about 20 minutes and 45 minutes, or between about 20 minutes and about 35 minutes, or between about 20 minutes and about 30 minutes, or between about 20 minutes and about 25 minutes, or between about 10 minutes and about 40 minutes, or between about 15 minutes and about 40 minutes, or between about 25 minutes and about 40 minutes, or between about 30 minutes and about 40 minutes, or between about 35 minutes and about 40 minutes.

[0084] In some aspects of the aforementioned method, step (a) may include incubating the mixture for at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes. In some aspects of the aforementioned method, step (a) may include incubating the mixture for about 10 minutes, or about 15 minutes, or about 20 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes.

[0085] In some aspects of the aforementioned methods, step (a) may include incubating the mixture at about room temperature (e.g., about 20° C.) for any period of time described above. In some aspects of the aforementioned methods, step (a) may include incubating the mixture at a temperature of about 18° C. to about 22° C. for any period of time described above.

[0086] Thus, in some aspects of the aforementioned methods, step (a) may comprise incubating the mixture at about 20°C for about 20 to about 40 minutes. Thus, in some aspects of the aforementioned methods, step (a) may comprise incubating the mixture at about 20°C for about 20 minutes.

[0087] In some aspects of the foregoing methods, separating the precipitate from the supernatant in step (c) can be accomplished using any method known in the art to separate the precipitate from the supernatant, including but not limited to manually removing the supernatant from the precipitate using a pipette, capillary, microtube, micropipette, or any combination thereof; or removing the supernatant from the precipitate using an automated liquid handling device, wherein the automated liquid handling device uses a pipette, capillary, microtube, micropipette, or any combination thereof to aspirate the supernatant from the precipitate.

[0088] In some aspects of the foregoing methods, step (d) may comprise incubating the resuspended pellet for between about 10 minutes and about 50 minutes, or between about 15 minutes and about 45 minutes, or between about 20 and about 40 minutes, or between about 25 and about 35 minutes, or between about 20 and about 50 minutes, or between about 20 minutes and about 45 minutes, or between about 20 minutes and about 35 minutes, or between about 20 minutes and about 30 minutes, or between about 20 minutes and about 25 minutes, or between about 10 minutes and about 40 minutes, or between about 15 minutes and about 40 minutes, or between about 25 minutes and about 40 minutes, or between about 30 minutes and about 40 minutes, or between about 35 minutes and about 40 minutes.

[0089] In some aspects of the aforementioned method, step (d) may include incubating the resuspended pellet for at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes. In some aspects of the aforementioned method, step (d) may include incubating the resuspended pellet for about 10 minutes, or about 15 minutes, or about 20 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes.

[0090] In some aspects of the foregoing methods, step (d) can comprise incubating the resuspended pellet at about 37° C. In some aspects of the foregoing methods, step (d) can comprise incubating the resuspended pellet at about 35° C. to about 39° C.

[0091] Thus, in some aspects of the aforementioned methods, step (d) may comprise incubating the resuspended pellet at about 37° C. for between about 20 and about 40 minutes. Thus, in some aspects of the aforementioned methods, step (d) may comprise incubating the resuspended pellet at about 20° C. for about 20 minutes.

[0092] In some aspects of the aforementioned methods, the resuspension solution may comprise any solution known in the art that is compatible with the antibody-nucleic acid conjugate. In a non-limiting example, the resuspension solution may comprise phosphate buffered saline. In a non-limiting example, the resuspension solution may comprise at least one biological buffer (e.g., at least one of HEPES, MOPS, MES, BES, MOPSO, ACES, TAPS, Bicine, Tricine, or any other biological buffer known in the art). In some aspects, the resuspension solution may comprise at least one salt.

[0093] In some aspects of the foregoing methods, steps (a)-(d) can be repeated at least once or at least twice or at least three times or at least four times or at least five times or at least six times or at least seven times or at least eight times or at least nine times or at least ten times.

[0094] In some aspects of the foregoing methods, the purity of the antibody-nucleic acid conjugate of the purified solution after step (d) or after repeating steps (a)-(d) at least once may be at least about 90% or at least about 91% or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or at least about 99.5% as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0095] In some aspects of the foregoing methods, the purity of the antibody-nucleic acid conjugate of the purified solution after step (d) or after repeating steps (a)-(d) at least once may be about 90%, about 91%, about 92% or about 93% or about 94%, about 95% or about 96%, about 97%, about 98% or 99% or about 99.5%, as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0096] In some aspects of the foregoing methods, the antibody-nucleic acid conjugate purity of the purified solution after step (d) or after repeating steps (a)-(d) at least once can be between about 85% and about 95%, or between about 87.5% and about 97.5%, or between about 90% and about 99%, or between about 90% and about 95%, as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0097] In some aspects of the foregoing methods, the purity of the antibody-nucleic acid conjugate of the purified solution after repeating steps (a)-(d) at least once may be at least about 90% or at least about 91% or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or at least about 99.5%, as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0098] In some aspects, the purity as measured by SEC HPLC is calculated by determining the area under the 260 nm curve corresponding to the antibody-nucleic acid conjugate and dividing it by the sum of the area under the 260 nm curve corresponding to the antibody-nucleic acid conjugate and the area under the 260 nm curve corresponding to the free, unconjugated nucleic acid. That is, the purity can be calculated using the following formula:

[0099]

[0100] In some aspects of the foregoing methods, the yield of the antibody-nucleic acid conjugate is at least about 60% or at least about 62.5% or at least about 65% or at least about 67.5% or at least about 70% or at least about 72.5% or at least about 75% or at least about 77.5% or at least about 80% or at least about 82.5% or at least about 85% or at least about 87.5% or at least about 90% or at least about 92.5% or at least about 95% or at least about 97.5% or at least about 99% or at least about 99.5%. In some aspects of the foregoing methods, the yield of the antibody-nucleic acid conjugate is 60% or 62.5% or 65% or 67.5% or 70% or 72.5% or 75% or 77.5% or 80% or 82.5% or 85% or 87.5% or 90% or 92.5% or 95% or 97.5% or 99% or 99.5%. In some aspects of the foregoing methods, the yield of the antibody-nucleic acid conjugate is at least about 80%.

[0101] As the skilled artisan will appreciate, the yield can be calculated by measuring the amount of antibody-nucleic acid conjugate before and after purification and then applying the following formula:

[0102]

[0103] The amount of antibody-nucleic acid conjugate can be measured using any method known in the art to quantify the amount of antibody-nucleic acid conjugate.

[0104] As the skilled artisan will appreciate, the yield can be calculated by measuring the amount of antibody (ie, the amount of protein) before and after purification, and then applying the following formula:

[0105]

[0106] In embodiments where the conjugation reaction is performed prior to purification of the antibody-nucleic acid conjugate, the yield can be calculated by measuring the amount of antibody input into the conjugation reaction (i.e., the amount of protein) and the amount of antibody obtained after purification, and then applying the following formula:

[0107]

[0108] The amount of antibody can be measured using any method known in the art to quantify the amount of protein, including but not limited to Bradford protein assay, UV-visible spectroscopy, biuret protein assay, Lowry protein assay, BCA protein assay, amidomelanin assay, and colloidal gold protein assay.

[0109] Methods for producing antibody-nucleic acid conjugates

[0110] The present disclosure provides a method for producing an antibody-nucleic acid conjugate comprising the following: i) contacting a first solution comprising a nucleic acid comprising at least one reactive group with a second solution comprising an antibody under conditions such that at least one reactive group of the nucleic acid reacts with the antibody to form an antibody-nucleic acid conjugate; ii) purifying the antibody-nucleic acid conjugate using any purification method described herein. In some aspects of the foregoing method, the buffer exchange step may be performed on one or more of the solutions before step (i) or after step (i) and before step (ii), using any buffer exchange procedure known in the art (e.g., spin filter buffer exchange, PD-10 column, etc.). A skilled technician will recognize and readily determine the conditions under which at least one reactive group of the nucleic acid reacts with the antibody to form an antibody-nucleic acid conjugate.

[0111] In some aspects of the aforementioned methods, the reactive group can be any reactive group known in the art that can be used for covalently linking nucleic acid molecules and protein molecules (e.g., antibodies). Non-limiting examples of reactive groups include, but are not limited to, maleimide, NHS ester, azide, alkyne, DBCOs, and tetrazine. Therefore, at least one reactive group includes at least one selected from the following parts: maleimide moiety, iodoacetamide moiety, benzyl halide moiety, bromomethyl ketone moiety, isothiocyanate moiety, succinimide ester moiety, carboxylate moiety, sulfosuccinimide ester moiety, 4-sulfotetrafluorophenyl (STP) ester moiety, 2,4,5,6-tetrafluorophenyl (TFP) ester moiety, sulfodichlorophenol (SDP) ester moiety, carbonyl azide moiety, N-hydroxysuccinimide (NHS) moiety, azide moiety, alkyne moiety, tetrazine moiety, aza-dibenzocyclooctyne moiety, and sulfonyl chloride moiety.

[0112] As will be appreciated by the skilled artisan, antibody-nucleic acid conjugates can be formed by a labeling reaction in which a nucleic acid comprising one or more reactive groups reacts with one or more amino acid side chains and / or portions of a polypeptide backbone and forms a covalent bond. These amino acid side chains may include, but are not limited to, cysteine ​​side chains, lysine side chains, or any other reactive side chains known in the art.

[0113] In aspects where the antibody-nucleic acid conjugate is formed via a cysteine ​​labeling reaction (eg, the nucleic acid comprises a maleimide-reactive group), prior to step (i), the antibody may be treated with a reducing agent (eg, TCEP, DTT, or any other reducing agent known in the art).

[0114] In some aspects of the methods for producing antibody-nucleic acid conjugates described herein, the conjugation step can be performed in a multi-well plate, wherein each well contains the same or different antibodies and / or nucleic acids. Subsequently, the antibody-nucleic acid conjugates produced in different wells can be combined and purified together before purification using the methods described herein to produce a plurality of antibody-nucleic acid conjugates comprising a plurality of different types of antibody-nucleic acid conjugates.

[0115] Antibody-nucleic acid conjugates of the present disclosure

[0116] As used herein, the term "antibody-nucleic acid conjugate" is used in the broadest sense to refer to a molecule comprising one or more antibodies (described in further detail herein) covalently linked to one or more nucleic acids (described in further detail herein). In a non-limiting example, an antibody-nucleic acid conjugate may comprise a single antibody covalently linked to a single nucleic acid.

[0117] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity. An antibody that binds to a target refers to an antibody that is capable of binding to a target with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting the target.

[0118] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-domain antibodies (sdAb); Camelid VHH domains; and multispecific antibodies formed from antibody fragments.

[0119] As will be appreciated by the skilled artisan, antibodies can be specifically designed and / or modified at one or more specific sites of the antibody to react with nucleic acids comprising reactive groups. In one non-limiting example, an antibody can be designed with a single exposed cysteine ​​that can react with nucleic acids comprising a maleimide group to form an antibody-nucleic acid conjugate.

[0120] Nucleic acids of the present disclosure

[0121] As used herein, the term "nucleic acid" is used in the broadest sense to refer to molecules comprising one or more polynucleotide chains.

[0122] As used herein, the term "polynucleotide" is used in the broadest sense to describe a polymer comprising two or more nucleotide monomers covalently bonded in a chain.

[0123] The nucleic acids of the present disclosure may be single-stranded (i.e., composed of a single polynucleotide chain), double-stranded (i.e., composed of two polynucleotide chains hybridized together by Watson-Crick or non-Watson base pairing), or partially double-stranded (i.e., having both single-stranded and double-stranded regions). The nucleic acids of the present disclosure may comprise any number of polynucleotide chains. The polynucleotide chains that are hybridized together may be "perfectly matched" such that the polynucleotide chains form a double-stranded structure with each other, such that each nucleotide in each chain undergoes Watson-Crick base pairing with a nucleotide in the other chain. The hybridized polynucleotide chains may comprise at least one mismatch, wherein the term "mismatch" means that a pair of nucleotides in the duplex cannot undergo Watson-Crick bonding.

[0124] The nucleic acid of the present disclosure may include at least one natural base. The nucleic acid of the present disclosure may not include a natural base. The nucleic acid of the present disclosure may include at least one modified nucleotide or nucleic acid analog. The nucleic acid of the present disclosure may not include a modified nucleotide or nucleic acid analog. The nucleic acid of the present disclosure may include at least one universal base. The nucleic acid of the present disclosure may not include a universal base.

[0125] The nucleic acids of the present disclosure may comprise any combination of natural bases (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural bases), modified nucleotides or nucleic acid analogs (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides or nucleic acid analogs), and / or universal bases (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more universal bases). When present in combination, the natural bases, modified nucleotides or nucleic acid analogs, and / or universal bases of the nucleic acid may be arranged in any order.

[0126] The term "modified nucleotide" or "nucleic acid analog" includes, but is not limited to, locked nucleic acid (LNA), bridged nucleic acid (BNA), propyne-modified nucleic acid, zipper nucleic acid Isoguanine, isocytosine 6-amino-1-(4-hydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one (PPG) and 2'-modified nucleic acids, such as 2'-O-methyl nucleic acids. The target binding domain may include 0-6 (e.g., 0, 1, 2, 3, 4, 5 or 6) modified nucleotides or nucleic acid analogs. Preferably, the modified nucleotide or nucleic acid analog is a locked nucleic acid (LNA).

[0127] The term "locked nucleic acid" as used herein includes, but is not limited to, modified RNA nucleotides in which the ribose moiety comprises a methylene bridge connecting the 2' oxygen and the 4' carbon. The methylene bridge locks the ribose in a 3'-endo conformation, also known as the North conformation, which is present in an A-type RNA duplex. The term inaccessible RNA can be used interchangeably with LNA. The term "bridged nucleic acid (BNA)" as used herein includes, but is not limited to, a modified RNA molecule comprising a five-membered or six-membered bridging structure with a fixed 3'-endo conformation, also known as the North conformation. The bridging structure connects the 2' oxygen of the ribose to the 4' carbon of the ribose. Various different bridge structures may contain carbon, nitrogen and hydrogen atoms. The term "propyne-modified nucleic acids" as used herein includes, but is not limited to, pyrimidines containing propyne modifications at the C5 position of the nucleic acid base, i.e., cytosine and thymine / uracil. The term "zipper nucleic acid" as used herein " includes, but is not limited to, polynucleotides conjugated to a cationic spermine moiety.

[0128] The term "universal base" as used herein includes, but is not limited to, nucleotide bases that do not follow the Watson-Crick base pairing rules but can bind to any of the four classical bases (A, T / U, C, G) located on the target nucleic acid.

[0129] In some aspects, the nucleic acids of the present disclosure may comprise at least one cleavable portion. Exemplary cleavable portions include, but are not limited to, chemical cleavable portions (e.g., portions that are cleaved when exposed to a particular chemical, combination of chemicals, or reaction conditions), photocleavable portions (e.g., portions that are cleaved when exposed to light of a sufficient wavelength or light containing a sufficient range of wavelengths), or enzymatic cleavable portions (e.g., portions that are cleaved by a particular enzyme or class of enzymes).

[0130] In some aspects, the nucleic acids of the present disclosure may comprise, consist essentially of, or consist of deoxyribonucleic acid (DNA). In some aspects, the nucleic acids of the present disclosure may comprise, consist essentially of, or consist of ribonucleic acid (RNA). In some aspects, the nucleic acids of the present disclosure may comprise, consist essentially of, or consist of a combination of DNA and RNA.

[0131] In some aspects, the DNA can be D-DNA (dextrorotatory). Thus, in some aspects, the nucleic acids of the present disclosure can comprise, consist essentially of, or consist of D-DNA.

[0132] In some aspects, the DNA can be L-DNA (left-handed). Thus, in some aspects, the nucleic acids of the present disclosure can comprise, consist essentially of, or consist of L-DNA.

[0133] In some aspects, the length of a nucleic acid of the present disclosure can be at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25. or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, or at least about 31, or at least about 32, or at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40, or at least about 41, or at least about 42, or at least about 43, or at least about 44, or at least about 45, or at least about 46, or at least about 47, or at least about 48, or at least about 49, or at least about 50. or at least about 51 or at least about 52 or at least about 53 or at least about 54 or at least about 55 or at least about 56 or at least about 57 or at least about 58 or at least about 59 or at least about 60 or at least about 61 or at least about 62 or at least about 63 or at least about 64 or at least about 65 or at least about 66 or at least about 67 or at least about 68 or at least about 69 or at least about 70 or at least about 71 or at least about 72 or at least about 73 or at least about 74 or at least about 75 or at least about 76 or at least about 77 or at least about 78 or at least about 79 or at least about 80 or at least about 81 or at least about 82 or at least about 83 or at least about 84 or at least about 85 or at least about 86 or at least about 87 or at least about 88 or at least about 89 or at least about 90 or at least about 91 or at least about 92 or at least about 93 or at least about 94 or at least about 95 or at least about 96 or at least about 97 or at least about 98 or at least about 99 or at least about 100 or more. At least about 76, or at least about 77, or at least about 78, or at least about 79, or at least about 80, or at least about 81, or at least about 82, or at least about 83, or at least about 84, or at least about 85, or at least about 86, or at least about 87, or at least about 88, or at least about 89, or at least about 90, or at least about 91, or at least about 92, or at least about 93, or at least about 94, or at least about 95, or at least about 96, or at least about 97, or at least about 98, or at least about 99, or at least about 100 nucleotides.

[0134] In some aspects, the length of a nucleic acid of the present disclosure can be about 1 or about 2, or about 3 or about 4 or about 5 or about 6 or about 7 or about 8 or about 9 or about 10 or about 11 or about 12 or about 13 or about 14 or about 15 or about 16 or about 17 or about 18 or about 19 or about 20 or about 21 or about 22 or about 23 or about 24 or about 25. about 25 or about 26 or about 27 or about 28 or about 29 or about 30 or about 31 or about 32 or about 33 or about 34 or about 35 or about 36 or about 37 or about 38 or about 39 or about 40 or about 41 or about 42 or about 43 or about 44 or about 45 or about 46 or about 47 or about 48 or about 49 or about 50 or about 51 or about 52 or about 53 or about 54 or about 55 or about 56 or about 57 or about 58 or about 59 or about 60 or about 61 or about 62 or about 63 or about 64 or about 65 or about 66 or about 67 or about 68 or about 69 or about 70 or about 71 or about 72 or about 73 or about 74 or about 75 or about 76 or about 77 or about 78 or about 79 or about 80 or about 81 or about 82 or about 83 or about 84 or about 85 or about 86 or about 87 or about 88 or about 89 or about 90 or about 91 or about 92 or about 93 or about 94 or about 95 or about 96 or about 97 or about 98 or about 99 or about 100 nucleotides.

[0135] In some aspects, the disclosed nucleic acids may be greater than about 100 nucleotides in length.

[0136] In some aspects, the length of the nucleic acids of the present disclosure can be about 60 to about 70 nucleotides. In some aspects, the length of the nucleic acids of the present disclosure can be about 66 nucleotides.

[0137] In some aspects, the length of the nucleic acids of the present disclosure can be about 50 to about 60 nucleotides. In some aspects, the length of the nucleic acids of the present disclosure can be about 56 nucleotides.

[0138] Kits of the present disclosure

[0139] The present disclosure provides kits for use in the methods described herein.

[0140] The present disclosure provides a kit comprising a purification solution including PEG and NaCl.

[0141] In some aspects, the purified solution may include a concentration of about 30% or about 31% or about 32% or about 33% or about 34% or about 35% or about 36% or about 37% or about 38% or about 39% or about 40% or about 41% or about 42% or about 43% or about 44% or about 45% or about 46% or about 47% or about 48% or about 49% or about 50% (w / v) PEG. In some aspects, the purified solution may include a concentration of about 30%-about 50% or about 35%-about 45% PEG. In some aspects, the purified solution may include a concentration of about 40% PEG.

[0142] As will be appreciated by those skilled in the art, polyethylene glycol (PEG) is a polymer of ethylene oxide. PEG can be obtained with a variety of different molecular weights based on the length of the polymer. Therefore, in some aspects of the aforementioned method, PEG can be PEG-100, PEG-200, PEG-300, PEG-400, PEG-500, PEG-600, PEG-700, PEG-800, PEG-900, PEG-1000, PEG-1500, PEG-2000, PEG-3000, PEG-3350, PEG-4000, PEG-5000, PEG-6000, PEG-7000, PEG-8000, PEG-9000, PEG-10000, PEG-15000, PEG-20000 or PEG-35000. In some aspects, PEG can be PEG-6000. In some aspects, PEG can be PEG-8000.

[0143] In some aspects, the purified solution may include a concentration of about 150mM or about 155mM or about 160mM or about 165mM or about 170mM or about 175mM or about 180mM or about 185mM or about 190mM or about 195mM or about 200mM or about 205mM or about 210mM ​​or about 215mM or about 220mM or about 225mM or about 230mM or about 235mM or about 240mM or about 245mM or about 250mM NaCl. In some aspects, the purified solution may include a concentration of about 150mM-about 250mM or about 175mM-about 225mM NaCl. In some aspects, the purified solution may include a concentration of about 200mM NaCl.

[0144] Thus, in some aspects, the purification solution may comprise a PEG concentration between about 30% and about 50% (w / v) and a NaCl concentration between about 150 mM and about 250 mM. In some aspects, the purification solution may comprise a PEG concentration of about 40% (w / v) and a NaCl concentration of about 200 mM.

[0145] In some aspects, the kit may further comprise at least one solution containing the disclosed nucleic acid, wherein the nucleic acid comprises at least one reactive group. The reactive group may be any reactive group known in the art that can be used for covalently linking nucleic acid molecules and protein molecules (e.g., antibodies). Non-limiting examples of reactive groups include, but are not limited to, maleimide, NHS ester, azide, alkyne, DBCOs, and tetrazine.

[0146] In some aspects, the kit may further comprise a first solution containing a nucleic acid of the present disclosure, wherein the nucleic acid comprises at least one reactive group and at least a second solution containing a nucleic acid of the present disclosure, wherein the nucleic acid comprises at least one reactive group, wherein the nucleic acid in the first solution comprises a nucleic acid sequence that is completely or at least partially different from the nucleic acid sequence of the nucleic acid in the at least second solution.

[0147] In some aspects, the kit can further comprise a solution comprising at least one reducing agent (eg, TCEP, DTT, or any other reducing agent known in the art).

[0148] In some aspects, the kit can further comprise a solution comprising an antibody of the disclosure.

[0149] In some aspects, the kit can further comprise at least one spin filter.

[0150] In some aspects, the kit can further comprise instructions for using the kit to perform at least one method described herein. In some aspects, the instructions can be written instructions.

[0151] Exemplary embodiments

[0152] Embodiment 1. A method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising:

[0153] a) contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG, and NaCl, such that the concentration of PEG in the mixture is between about 5% and about 30% (w / v) and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM;

[0154] b) centrifuging the mixture to produce a supernatant and a precipitate;

[0155] c) separating the precipitate from the supernatant;

[0156] d) resuspending the pellet with a resuspending solution to produce a purified solution;

[0157] e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution having a purity of at least 90% for the antibody-nucleic acid conjugate as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0158] Embodiment 2. The method of embodiment 1, wherein the concentration of PEG in the mixture is between about 10% and about 25% (w / v).

[0159] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the concentration of NaCl in the mixture is between about 150 mM and about 200 mM.

[0160] Embodiment 4. The method of any of the preceding embodiments, wherein the antibody-nucleic acid conjugate purity of the purified solution is at least 95% as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

[0161] Embodiment 5. The method of any of the preceding embodiments, wherein step (b) comprises centrifuging the mixture at a speed of at least about 1,000 RCF (xg),

[0162] Embodiment 6. The method of any of the preceding embodiments, wherein the centrifugation in step (b) is performed for at least about 2 minutes.

[0163] Embodiment 7. The method of any of the preceding embodiments, wherein step (a) further comprises incubating the mixture at about 20°C for at least about 20 to about 40 minutes.

[0164] Embodiment 8. The method of any of the preceding embodiments, wherein step (d) further comprises incubating the resuspended pellet at about 37°C for about 20 to about 40 minutes.

[0165] Embodiment 9. The method of any of the preceding embodiments, wherein step (a) further comprises vortexing the mixture.

[0166] Embodiment 10. The method of any one of the preceding embodiments, wherein the resuspension solution comprises phosphate buffered saline (PBS).

[0167] Embodiment 11. The method of any one of the preceding embodiments, wherein PEG is:

[0168] i) PEG-6000;

[0169] ii) PEG-8000;

[0170] iii) PEG-10000; or

[0171] iv) PEG-35,000.

[0172] Embodiment 12. The method of embodiment 11, wherein the PEG is PEG-6000.

[0173] Embodiment 13. The method of embodiment 11, wherein the PEG is PEG-8000.

[0174] Embodiment 14. The method of any of the preceding embodiments, wherein contacting the solution with a purification solution in step (a) comprises contacting the solution with the purification solution in a ratio of 1:1 (v / v).

[0175] Embodiment 15. The method of any of the preceding embodiments, wherein the concentration of PEG in the purification solution is between about 35% and about 45% (w / v), preferably wherein the concentration of PEG in the purification solution is about 40% (w / v).

[0176] Embodiment 16. The method of any one of the preceding embodiments, wherein the concentration of NaCl in the purification solution is between about 150 mM-250 mM, preferably wherein the concentration of NaCl in the purification solution is about 200 mM.

[0177] Embodiment 17. The method of any of the preceding embodiments, wherein the antibody comprises Fab, Fab', F(ab')2, Fv fragment, single-chain (sc)Fv (scFv) antibody fragment, linear antibody, single domain antibody (sdAb), Camelidae VHH domain, multispecific antibody or any combination thereof.

[0178] Embodiment 18. The method of any one of the preceding embodiments, wherein the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0179] Embodiment 19. The method of any one of the preceding embodiments, wherein the nucleic acid comprises L-DNA.

[0180] Embodiment 20. The method of any of the preceding embodiments, wherein the method is performed in one or more wells of a multi-well plate, thereby purifying multiple antibody-nucleic acid conjugates simultaneously.

[0181] Embodiment 21. The method of any of the preceding embodiments, wherein the method is performed in two or more wells of a multi-well plate, thereby purifying multiple antibody-nucleic acid conjugates in different wells simultaneously.

[0182] Embodiment 22. A method for producing an antibody-nucleic acid conjugate, the method comprising:

[0183] a) contacting a first solution comprising a nucleic acid including at least one reactive group with a second solution comprising an antibody under conditions such that the at least one reactive group of the nucleic acid reacts with the antibody, thereby forming an antibody-nucleic acid conjugate;

[0184] b) purifying the antibody-nucleic acid conjugate using the method of any one of embodiments 1-21.

[0185] Embodiment 23. The method of Embodiment 22, wherein at least one reactive group comprises a maleimide moiety.

[0186] Embodiment 24. The method of embodiment 22 or embodiment 23, wherein at least one reactive group comprises at least one moiety selected from the group consisting of a maleimide moiety, an iodoacetamide moiety, a benzyl halide moiety, a bromomethyl ketone moiety, an isothiocyanate moiety, a succinimide ester moiety, a carboxylate moiety, a sulfosuccinimide ester moiety, a 4-sulfotetrafluorophenyl (STP) ester moiety, a 2,4,5,6-tetrafluorophenyl (TFP) ester moiety, a sulfodichlorophenol (SDP) ester moiety, a carbonyl azide moiety, an N-hydroxysuccinimide (NHS) moiety, an azide moiety, an alkyne moiety, a tetrazine moiety, an aza-dibenzocyclooctyne moiety, and a sulfonyl chloride moiety.

[0187] Embodiment 25. A kit comprising:

[0188] i) a first solution comprising a nucleic acid comprising at least one reactive group; and

[0189] ii) A purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl).

[0190] Embodiment 26. The kit of embodiment 25, wherein the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0191] Embodiment 27. The kit of any one of Embodiments 25-26, wherein the nucleic acid comprises L-DNA.

[0192] Embodiment 28. The kit of any one of Embodiments 25-27, wherein at least one reactive group comprises a maleimide moiety.

[0193] Embodiment 29. The method of any one of Embodiments 25-27, wherein at least one reactive group comprises at least one moiety selected from the group consisting of a maleimide moiety, an iodoacetamide moiety, a benzyl halide moiety, a bromomethyl ketone moiety, an isothiocyanate moiety, a succinimide ester moiety, a carboxylate moiety, a sulfosuccinimide ester moiety, a 4-sulfotetrafluorophenyl (STP) ester moiety, a 2,4,5,6-tetrafluorophenyl (TFP) ester moiety, a sulfodichlorophenol (SDP) ester moiety, a carbonyl azide moiety, an N-hydroxysuccinimide (NHS) moiety, an azide moiety, an alkyne moiety, a tetrazine moiety, an aza-dibenzocyclooctyne moiety, and a sulfonyl chloride moiety.

[0194] Embodiment 30. The kit of any one of embodiments 25-29, further comprising a second solution comprising an antibody.

[0195] Embodiment 31. The kit of embodiment 30, wherein the antibody comprises Fab, Fab', F(ab')2, Fv fragment, single-chain (sc)Fv (scFv) antibody fragment, linear antibody, single domain antibody (sdAb), Camelidae VHH domain, multispecific antibody or any combination thereof.

[0196] Embodiment 32. The kit of any one of embodiments 25-31, further comprising a third solution comprising a nucleic acid comprising at least one reactive group, wherein the nucleic acid in the first solution comprises a nucleic acid sequence different from the nucleic acid sequence of the nucleic acid in the third solution.

[0197] Embodiment 33. The kit of any one of Embodiments 25-32, wherein PEG is:

[0198] i) PEG-6000;

[0199] ii) PEG-8000;

[0200] iii) PEG-10000; or

[0201] iv) PEG-35,000.

[0202] Embodiment 34. The kit of embodiment 33, wherein the PEG is PEG-6000.

[0203] Embodiment 35. The kit of embodiment 33, wherein the PEG is PEG-8000.

[0204] Embodiment 36. The kit of any one of Embodiments 25-35, wherein the concentration of PEG in the purification solution is between about 35% and about 50% (w / v), preferably wherein the concentration of PEG in the purification solution is about 40% (w / v).

[0205] Embodiment 37. The kit of any one of Embodiments 25-36, wherein the concentration of NaCl in the purification solution is between about 150 mM-250 mM, preferably wherein the concentration of NaCl in the purification solution is about 200 mM.

[0206] Embodiment 38. The kit of any one of Embodiments 25-37, further comprising at least one aliquot of TCEP.

[0207] Embodiment 39. The kit of any one of Embodiments 25-38, further comprising at least one spin filter.

[0208] Embodiment 40. The kit of any one of embodiments 25-39, further comprising at least one multi-well plate.

[0209] Example

[0210] Example 1 - Purification of Antibody-Nucleic Acid Conjugates Using the Disclosed Methods

[0211] The following is a non-limiting example that demonstrates that the purification method of the present disclosure can be used to purify an antibody-nucleic acid conjugate with a purity exceeding 90% from the unconjugated nucleic acid remaining from the conjugation reaction.

[0212] Antibody-nucleic acid conjugates were prepared using standard cysteine ​​labeling methods known in the art. Briefly, the antibodies were treated with the reducing agent TCEP, washed using a spin filter, and incubated with nucleic acids comprising reactive groups capable of reacting with cysteine ​​residues on the antibody. These antibody-nucleic acid conjugates were then purified using one of two separate protocols.

[0213] In the first scheme, the conjugation reaction comprising the antibody-nucleic acid conjugate and the unconjugated nucleic acid is contacted with a purification solution comprising PEG-6000 to produce a mixture comprising the antibody-nucleic acid conjugate, the unconjugated nucleic acid and PEG, such that the concentration of PEG in the mixture is about 25% (w / v). The mixture is then centrifuged to produce a supernatant and a precipitate, the precipitate is separated from the supernatant, and the precipitate is resuspended using a resuspension solution comprising PBS.

[0214] In the second scheme, the conjugation reaction comprising the antibody-nucleic acid conjugate and the unconjugated nucleic acid is contacted with a purification solution comprising PEG-6000 and NaCl to produce a mixture comprising the antibody-nucleic acid conjugate, the unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is about 25% (w / v) and the concentration of NaCl is about 100 mM. The mixture is then centrifuged to produce a supernatant and a precipitate, the precipitate is separated from the supernatant, and the precipitate is resuspended using a resuspension solution comprising PBS.

[0215] The purified solution was then analyzed using SEC HLPC to determine the extent of purification of the antibody-nucleic acid conjugate.

[0216] As a first control, unconjugated antibody was analyzed using SEC HLPC. The results of this analysis are shown in Figure 1 shown. Figure 1 The horizontal lines in the graph represent, from left to right, the expected elution times of the antibody-nucleic acid conjugate, unconjugated nucleic acid, and unconjugated antibody.

[0217] As a second control, an unpurified conjugation reaction was analyzed using SEC HLPC. The results of this analysis are shown in Figure 2 shown. Figure 2 The horizontal lines in the graph represent the expected elution times of antibody-nucleic acid conjugate, unconjugated nucleic acid, and unconjugated antibody from left to right. As expected, the unpurified conjugation reaction showed an excess of unconjugated nucleic acid.

[0218] The conjugation reaction purified using the first purification scheme described above was analyzed using SEC HPLC. The results of this analysis are shown in Figure 3 shown. Figure 3 The horizontal lines in the graph from left to right indicate the expected elution times of the antibody-nucleic acid conjugate, unconjugated nucleic acid, and unconjugated antibody. Figure 3 As shown, the first purification scheme resulted in little or no purification of the antibody-nucleic acid conjugate.

[0219] The conjugation reaction purified using the second purification scheme described above was analyzed using SEC HPLC. The results of this analysis are shown in Figure 4 shown. Figure 4 The horizontal lines in the graph from left to right indicate the expected elution times of the antibody-nucleic acid conjugate, unconjugated nucleic acid, and unconjugated antibody. Figure 4 As shown, the second purification scheme produced purified antibody-nucleic acid conjugates with a purity of over 90% from contaminating unconjugated nucleic acids.

[0220] Thus, the results described in this Example demonstrate that the purification methods of the present disclosure, and more specifically the use of PEG and NaCl at the concentrations described herein, allow for the purification of antibody-nucleic acid conjugates to high levels of purity, including levels exceeding 90%.

[0221] Example 2 - Purification of Antibody-Nucleic Acid Conjugates Using the Disclosed Methods

[0222] The following is a non-limiting example that demonstrates that the purification method of the present disclosure can be used to purify an antibody-nucleic acid conjugate.

[0223] Antibody-nucleic acid conjugates were prepared as described in Example 1. These antibody-nucleic acid conjugates were then purified using one of two separate protocols.

[0224] In the first scheme, the conjugation reaction containing the antibody-nucleic acid conjugate and the unconjugated nucleic acid is contacted with a purification solution containing PEG-6000 and NaCl to produce a mixture containing the antibody-nucleic acid conjugate, the unconjugated nucleic acid, PEG and NaCl, so that the concentration of PEG in the mixture is about 25% (w / v) and the concentration of NaCl is about 100 mM. The mixture is then centrifuged to produce a supernatant and a precipitate, the precipitate is separated from the supernatant, and the precipitate is resuspended using a resuspension solution containing PBS. These steps are then repeated twice to produce a purified solution of the antibody-nucleic acid conjugate.

[0225] In the second scheme, the conjugation reaction containing the antibody-nucleic acid conjugate and the unconjugated nucleic acid is contacted with a purification solution containing PEG-6000 and NaCl to produce a mixture containing the antibody-nucleic acid conjugate, the unconjugated nucleic acid, PEG and NaCl, so that the concentration of PEG in the mixture is about 25% (w / v) and the concentration of NaCl is about 200 mM. The mixture is then centrifuged to produce a supernatant and a precipitate, the precipitate is separated from the supernatant, and the precipitate is resuspended using a resuspension solution containing PBS. These steps are then repeated twice to produce a purified solution of the antibody-nucleic acid conjugate.

[0226] The solutions produced by each of the two purification schemes described above, as well as aliquots of the unpurified conjugation reactions, were then analyzed using SEC HLPC to determine the extent of purification of the antibody-nucleic acid conjugation reactions. The results of this analysis are shown in Table 1. Figure 5 shown. Figure 5 The horizontal lines in the graph from left to right indicate the expected elution times of the antibody-nucleic acid conjugate, unconjugated nucleic acid, and unconjugated antibody. Figure 5 As shown, purification of the antibody-nucleic acid conjugate was achieved using both protocols.

[0227] Therefore, the results described in this example show that the purification method of the present disclosure, and more specifically the use of PEG and NaCl at the concentrations described herein, allows the purification of antibody-nucleic acid conjugates to a high purity level. In addition, the results show that the concentration of NaCl can be varied within the range specified herein to optimize the overall purity of the antibody-nucleic acid conjugate.

[0228] Example 3 - Antibody-nucleic acid conjugates purified using the disclosed methods show unchanged activity

[0229] The following is a non-limiting example demonstrating that the purification method of the present disclosure does not alter the activity of the purified antibody-nucleic acid conjugate compared to the antibody-nucleic acid conjugate purified using the existing SEC HPLC method.

[0230] 14 different antibody-nucleic acid conjugates were purified using a standard SEC HPLC method or the PEG precipitation method described herein. The purified antibody-nucleic acid conjugates were then used for GeoMx assays performed on cell pellet arrays (see Merritt et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol. 2020 May; 38(5): 586-599 for information on GeoMx assays). The functional signals obtained in the assay using SEC HPLC-purified antibody-nucleic acid conjugates and PEG-purified antibody-nucleic acid conjugates were then compared. The comparison is as follows Figure 6 As shown. Figure 6 As shown, the signals between the two purification methods are strongly correlated, indicating that the methods of the present disclosure do not compromise the activity of the purified antibody-nucleic acid conjugates.

[0231] Example 4 - Antibody-nucleic acid conjugates purified on multi-well plates using the methods of the present disclosure

[0232] The following is a non-limiting example that demonstrates that the purification method of the present disclosure can be performed in a multi-well (eg, 96-well) plate, thereby allowing for rapid and simultaneous purification of several different species of antibody-nucleic acid conjugates.

[0233] 12 different kinds of antibody-nucleic acid conjugates were purified using the PEG precipitation method described herein. Each kind of antibody-nucleic acid conjugate contained different nucleic acid species and all kinds of universal polyclonal rabbit IgG antibodies. Each species was purified in a single well of a 96-well plate. Table A shows the % yield of each different antibody-nucleic acid conjugate.

[0234] Table A

[0235]

[0236]

[0237] Example 5 - Various Antibody-Nucleic Acid Conjugates Purified Using the Disclosed Methods

[0238] The following is a non-limiting example that demonstrates that the purification method of the present disclosure is robust and can be used to purify a variety of different antibody-nucleic acid conjugates, including those that differ in the identity of the antibody and oligonucleotide used.

[0239] 8 different kinds of antibody-nucleic acid conjugates were purified using the PEG precipitation method described herein. As shown in Table B, each kind of antibody-nucleic acid conjugate contained different nucleic acid species and different antibody species. Each species was purified in a sample tube. Table B shows the % yield of each different antibody-nucleic acid conjugate.

[0240] Table B

[0241] Antibody-nucleic acid conjugates % Yield Rat IgG2b-Oligonucleotide 1 76 Mouse IgG2b-Oligonucleotide 2 61 Rabbit IgG Clone A-Oligodeoxynucleotide 3 65 Rabbit IgG Clone B-Oligo 4 62 Rabbit IgG Clone C-Oligonucleotide 5 73 Rabbit IgG Clone D-Oligonucleotide 6 74 Rabbit IgG Clone E-Oligo 7 74 Mouse IgG1-Oligonucleotide 8 72 average 70

[0242] Example 6 - Pooled purified antibody-nucleic acid conjugates using the disclosed methods show unchanged activity

[0243] The following is a non-limiting example, which shows that the purification method of the present disclosure can be performed in a "separate" form or in a "combined" form, and the choice of form does not change the activity of the purified antibody-nucleic acid conjugate. In this way, multiple different types of antibody-nucleic acid conjugates can be combined and purified simultaneously using the method of the present disclosure.

[0244] More than 20 different kinds of antibody-nucleic acid conjugates were purified separately (i.e., each kind was purified in its own sample tube) or in a pooled form (i.e., more than 20 kinds were pooled in a single sample tube before purification) using the PEG precipitation method described herein. The antibody-nucleic acid conjugates were then used to evaluate two different cell lines, NK92 cells and HCC78 cells. The functional signals recorded using the separately purified or pooled purified conjugates were then compared. This comparison was Fig. 7A and 7B As shown. Fig. 7A and 7B As shown, the signals between the two purified forms are strongly correlated, indicating that the methods of the present disclosure can be used to purify different species of antibody-nucleic acid conjugates separately or to purify a pool of multiple different species of antibody-nucleic acid conjugates.

Claims

1. A method for purifying an antibody-nucleic acid conjugate from a solution comprising the antibody-nucleic acid conjugate and unconjugated nucleic acid, the method comprising: a) contacting the solution with a purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl) to produce a mixture comprising antibody-nucleic acid conjugate, unconjugated nucleic acid, PEG and NaCl, such that the concentration of PEG in the mixture is between about 5% and about 30% (w / v) and the concentration of NaCl in the mixture is between about 100 mM and about 250 mM; b) centrifuging the mixture to produce a supernatant and a precipitate; c) separating the precipitate from the supernatant; d) resuspending the precipitate with a resuspending solution to produce a purified solution; e) repeating steps (a)-(d) at least once using the purified solution of step (d) to produce a purified solution having a purity of at least 90% for the antibody-nucleic acid conjugate as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

2. The method of claim 1, wherein the concentration of PEG in the mixture is between about 10% and about 25% (w / v).

3. The method of claim 1 or claim 2, wherein the concentration of NaCl in the mixture is between about 150 mM and about 200 mM.

4. The method according to any one of the preceding claims, wherein the antibody-nucleic acid conjugate purity of the purified solution is at least 95% as measured by size exclusion (SEC) high performance liquid chromatography (HPLC).

5. The method according to any one of the preceding claims, wherein step (b) comprises centrifuging the mixture at a speed of at least about 1,000 RCF (xg), preferably wherein the centrifugation in step (b) is performed for at least about 2 minutes.

6. A method according to any one of the preceding claims, wherein: i) step (a) further comprises incubating the mixture at about 20° C. for about 20 to about 40 minutes; and / or ii) step (d) further comprises incubating the resuspended pellet at about 37°C for about 20 to about 40 minutes.

7. The method according to any one of the preceding claims, wherein PEG is: i) PEG-6000; ii) PEG-8000; iii) PEG-10000; or iv) PEG-35,000.

8. The method according to any one of the preceding claims, wherein contacting the solution with a purification solution in step (a) comprises contacting the solution with the purification solution in a ratio of 1:1 (v / v).

9. The method according to any one of the preceding claims, wherein the concentration of PEG in the purification solution is between about 35% and about 45% (w / v), preferably wherein the concentration of PEG in the purification solution is about 40% (w / v).

10. The method according to any one of the preceding claims, wherein the concentration of NaCl in the purification solution is between about 150 mM and 250 mM, preferably wherein the concentration of NaCl in the purification solution is about 200 mM.

11. The method according to any one of the preceding claims, wherein the antibody comprises a Fab, a Fab', a F(ab')2, a Fv fragment, a single-chain (sc)Fv (scFv) antibody fragment, a linear antibody, a single domain antibody (sdAb), a Camelidae VHH domain, a multispecific antibody or any combination thereof.

12. The method according to any one of the preceding claims, wherein the method is performed in one or more wells of a multi-well plate, thereby purifying multiple antibody-nucleic acid conjugates simultaneously.

13. The method according to any one of the preceding claims, wherein the method is performed in two or more wells of a multiwell plate, thereby purifying multiple antibody-nucleic acid conjugates in different wells simultaneously.

14. A method for producing an antibody-nucleic acid conjugate, the method comprising: a) contacting a first solution comprising a nucleic acid including at least one reactive group with a second solution comprising an antibody under conditions such that the at least one reactive group of the nucleic acid reacts with the antibody, thereby forming an antibody-nucleic acid conjugate; b) purifying the antibody nucleic acid conjugate using the method according to any one of claims 1-14.

15. The method of claim 14, wherein the at least one reactive group comprises a maleimide moiety, an iodoacetamide moiety, a benzyl halide moiety, a bromomethyl ketone moiety, an isothiocyanate moiety, a succinimide ester moiety, a carboxylate moiety, a sulfosuccinimide ester moiety, a 4-sulfotetrafluorophenyl (STP) ester moiety, a 2,4,5,6-tetrafluorophenyl (TFP) ester moiety, a sulfodichlorophenol (SDP) ester moiety, a carbonyl azide moiety, an N-hydroxysuccinimide (NHS) moiety, an azide moiety, an alkyne moiety, a tetrazine moiety, an aza-dibenzocyclooctyne moiety, a sulfonyl chloride moiety, or any combination thereof.

16. A kit comprising: i) a first solution comprising a nucleic acid comprising at least one reactive group; and ii) A purification solution comprising polyethylene glycol (PEG) and sodium chloride (NaCl).

17. The kit of claim 16, wherein the at least one reactive group comprises a maleimide moiety, an iodoacetamide moiety, a benzyl halide moiety, a bromomethyl ketone moiety, an isothiocyanate moiety, a succinimide ester moiety, a carboxylate moiety, a sulfosuccinimide ester moiety, a 4-sulfotetrafluorophenyl (STP) ester moiety, a 2,4,5,6-tetrafluorophenyl (TFP) ester moiety, a sulfodichlorophenol (SDP) ester moiety, a carbonyl azide moiety, an N-hydroxysuccinimide (NHS) moiety, an azide moiety, an alkyne moiety, a tetrazine moiety, an aza-dibenzocyclooctyne moiety, a sulfonyl chloride moiety, or any combination thereof.

18. The kit of claim 16 or claim 17, further comprising a second solution comprising an antibody.

19. The kit of claim 18, wherein the antibody comprises a Fab, a Fab', a F(ab')2, a Fv fragment, a single-chain (sc)Fv (scFv) antibody fragment, a linear antibody, a single domain antibody (sdAb), a Camelidae VHH domain, a multispecific antibody, or any combination thereof.

20. The kit according to any one of claims 16 to 19, further comprising a third solution comprising a nucleic acid containing at least one reactive group. The nucleic acid in the first solution includes a nucleic acid sequence different from the nucleic acid sequence of the nucleic acid in the third solution.

21. The kit according to any one of claims 16 to 20, wherein PEG is: i) PEG-6000; ii) PEG-8000; iii) PEG-10000; or iv) PEG-35,000.

22. The kit according to any one of claims 16 to 21, wherein the concentration of PEG in the purification solution is between about 35% and about 50% (w / v), preferably wherein the concentration of PEG in the purification solution is about 40% (w / v).

23. The kit according to any one of claims 16 to 22, wherein the concentration of NaCl in the purification solution is between about 150 mM and about 250 mM, preferably wherein the concentration of NaCl in the purification solution is about 200 mM.

24. The kit according to any one of claims 16 to 23, wherein the kit further comprises at least one of the following: i) at least one aliquot of TCEP, ii) at least one spin filter, and iii) at least one multiwell plate.

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