Patent Application
20240182586
Non-specific chemical methods, such as acylation of amines (e.g., lysine) or alkylation of thiols (e.g., cysteine), are widely used to construct protein conjugates for many applications. These techniques have several shortcomings. For example, the lack of site-specificity results in heterogenous products, as well as a synthetic route that is irreproducible and prone to side reactions. Glycoproteins are particularly sensitive to non-specific modifications because of the added complexity of the oligosaccharide chains (glycans) attached to amino acid sidechains of the protein. Site-specific conjugation of antibodies is reviewed by Sadiki et al., 2020. Unpredictable modifications at or near the functional domains of glycoproteins may significantly reduce the targeting activity and specificity of the glycoproteins. Changes is binding specificity of antibodies are described by Cunningham et al., 2021. Genetic engineering of glycoproteins can take a bottom-up approach by altering the primary sequence to provide target sites for site-specific modification. This genetic engineering approach has various shortcomings, such as laborious optimization and low yield, provides only a linear architecture, and does not retain the native primary sequence and conformation. The protein conformation can primarily determine the site occupancy of glycosylated residues. To support the glycoprotein conformation, genetic engineering also can take a top-down or combined approach by, for example, transfecting non-human mammalian cells to modify a recombinant or native glycoprotein using human glycosyltransferases. The transfection approach may provide a high yield, but requires laborious front end optimization and testing because the products are unpredictable. Methods are needed to construct glycoprotein conjugates that better preserve the function of the glycoprotein while providing site-specific modifications.
The present technology provides methods for making a site-specific modification of a glycoprotein. The methods can preserve the charge and glycoform of the glycoprotein. A targeted amino acid for a site-specific modification is accessed by trimming one or more glycans from the glycoprotein, while keeping a core glycan attached to the glycoprotein. The attached core glycan can optionally be utilized for modulation of the glycoprotein or can be reattached to one or more glycans or reagents for glycan engineering.
The technology can be further summarized by the following list of features.
1. A method for making a site-specific modification of a glycoprotein, the method comprising the steps of:
As used herein, the term “about” refers to a range of within plus or minus 10%, 5%, 1%, or 0.5% of the stated value.
As used herein, “consisting essentially of” allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with the alternative expression “consisting of” or “consisting essentially of”.
The present technology provides methods for making a site-specific modification of a glycoprotein. The site-specific methods do not require changing the glycoform or conformation of the glycoprotein. The site-specific methods can preserve the effector functions of a glycoprotein after the modifications. For example, specificity of antibodies can be preserved after performing the methods. The methods can be used to modulate the functions of glycoproteins, for example, to decrease non-specific binding of labeled antibody-based imaging agents to fragment-gamma receptors (FcγRs) on immune cells.
Approximately half of proteins typically expressed in a cell undergo glycosylation, which entails the post translational covalent addition of sugar moieties (glycans) to specific amino acids. However, few methods exist to site-specifically conjugate reagents to glycoproteins, especially without the need for genetic engineering. Glycoproteins, such as antibodies, are of particular interest because these proteins largely control immune response.
An example technique is using transglutaminase (TGase, EC 2.3.2.13) to modify glutamine sidechains on glycoproteins. The TGase enzyme catalyzes the formation of an isopeptide amide bond between an unsubstituted. sidechain amide of glutamine residue (as an acyl donor) and a nucleophilic amine substrate (as an acyl acceptor). To catalyze the isopeptide bond, the TGase must have access to at least one glutamine sidechain of the glycoprotein. Glycans on the glycoprotein can sterically hinder all access to glutamine by TGase.
The usefulness of TGase for conjugating reagents to glycoproteins is severely limited by the steric hindrance. One approach is to cleave the glycans from a glycoprotein before conjugating a reagent to a glutamine sidechain of the glycoprotein. Complete removal of the glycans should be avoided because the glycans are key to the specific function, structure, and immunogenicity of the glycoprotein in vivo.
For targeted site-specific modification of a glycoprotein, access to a sidechain of a target modification site amino acid can be completely hindered by glycans covalently attached to nearby amino acid sidechains. At the left side of
Despite having over 60 glutamine residues, native (non-modified) antibodies are poor substrates for TGase. In this context, native antibodies can be antibodies that are either recombinantly produced or naturally existing in various species whereby their primary amino acid sequence is conserved. The sequences of native antibodies, though, are largely identified and conserved, in particular the Fc regions. TGase can be applied to native antibodies after entire glycans have been cleaved from the native antibodies. Applied to IgG1, prior to transamidation (access to a glutamine residue by TGase), one has to reduce the steric hindrance from a nearby conserved N-glycan (e.g., asparagine, Asn297, IgG1). The removal of the steric hindrance by completely cleaving glycans from Asn297 is illustrated at the top of
In a previous report, an amidase (PNGase F, E.C. 3.5.1.52) is used to completely remove the N-glycan from Asn297 (Dennler, et al., 2014). During this removal, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297, IgG1,
The complete deglycosylation of Asn297, e.g., by PNGase F, has several disadvantages. First, deglycosylation transforms the Asn297 (an amide) into aspartic acid Asp297 (a carboxylic acid). This deamidation process results in a negatively charged group at physiological conditions. The charge variant leads to numerous issues such as significant structural changes, functional perturbation and immunogenicity, which ultimately may affect the function and efficacy of the antibody conjugates. This transformation results in a conformational change that may lead to decrease in stability in vivo, increase aggregation tendency, as well as abolishment of Fc-related biological activity such as effector function, for example, antibody dependent cellular cytotoxicity (ADCC).
Protein engineering is also reported and includes insertion or deletion of reactive glutamine(s) via mutagenesis or incorporation of peptide tags (with a reactive glutamine) onto the terminal ends of the antibody (Strop, et al., 2016; Anami, et al., 2017; Schneider, et al., 2020). These genetic engineering approaches have various shortcomings such as laborious optimization, low yield, provide only a linear architecture and do not retain the native primary sequence. Consequently, not all antibodies are amenable to the genetic route.
An example of the technology disclosed herein is illustrated at the bottom of
The example depicted at the bottom of
The methods herein enable tailoring of N-glycans, site specific-modification of the associated glycoprotein, and optional modulation of the glycoprotein. As depicted in
Naturally occurring or engineered enzymes such as Endoglycosidases (EndoS, E.C. 3.2.1.96) can be used to trim glycans on antibodies. Endo S is derived from Streptococcus pyogenes and includes a family of enzymes that catalyze a hydrolysis reaction of complex type N-linked glycans (e.g., innermost core N-Acetylglucosamine, GlcNAc) on macromolecules such as antibodies. EndoS exhibits endo-β-N-acetylglucosaminidase activity with catalytic glutamic residues and several tryptophan residues that play an important role in catalysis (Sjogren, et al., 2013). This enzyme displays a variety of substrate specificities. For example, EndoS hydrolyzes N-linked glycans in native antibodies and not denatured antibodies. Other examples of endoglycosidases include Endo S2, Endo H, Endo F1, Endo F2, Endo F3, Endo D, Endo M, O-glycosidase derived from Streptococcus oralis, O-glycosidase derived from Streptococcus pneumoniae, and O-glycosidase derived from Enterococcus faecalis (Fujita, et al., 2005; Koutsioulis, et al., 2008). EndoS is commercially available and applicable to a wide range of antibody isotypes including human IgGs 1-4, mouse IgGs 1 and 3, rabbit IgG, rat IgGs 1 and 2a, bovine IgGs 1 and 2, feline and canine IgGs, as well as equine IgGs 1-7 (e.g., Table 1). O-Glycosidase, also known as endo-α-N-acetylgalactosaminidase (EC 3.2.1.97), catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.
At the bottom of
The technology provides new methods for modification of antibodies and their related fragments via a chemoenzymatic process. The method includes trimming of native or engineered glycan(s) on antibodies and their related fragments, namely glycan remodeling, to render antibody's glutamine residue(s) accessible for conjugation by TGase. This can be followed by conjugation of a native or engineered glutamine residue(s) with an amine containing reagents or other suitable reagents via a transamidation reaction mediated by TGase.
The methods presented herein are proven to provide advantageous attributes. First, the endoglycosidase-hydrolysis (i.e., glycan remodeling or trimming) results in a single and homogenous glycoform. This construct can be less immunogenic than the previous PNGaseF hydrolysis that converts a conserved asparagine to an aspartic acid. Second, the glycan remodeling process is versatile by facile modulation of Fc-related biological activity, e.g., ADCC. Model antibodies are utilized herein. Both model antibodies utilized (cetuximab and Infliximab) have effector function. To completely abolish the effector function, EndoS2 can be utilized. The abolishment of the effector function results in better imaging agents by decreasing non-specific binding; for example, by decreasing binding of the Fc domain to Fcγ receptors on immune cells.
A facile process to build antibody-chromophore conjugates via convergent assembly is provided. This approach confers a higher binding efficiency than its non-specific conjugation equivalent. A new method to modify a native antibody using TGase while retaining the antibody's primary sequence and core glycan is also provided.
The technology can be applied to a variety of glycoproteins. The endoglycosidase can be used to trim an O-glycan linked to a sidechain of the serine or threonine residue. Trimming of the O-glycan causes a modification site amino acid residue to become accessible for attachment of a reagent. Sialic acids can be removed from the O-glycan using a sialidase.
The technology contemplates that as more endoglycosidases become available, the methods can be applied to a larger variety of glycoproteins. An schematic diagram of an embodiment of a method for making a site-specific modification of a glycoprotein is shown in
Trimming of an N-glycan or an O-glycan can also be performed using a chemical method instead of an endoglycosidase. An example of such a chemical method is reported by Sojar et al. (1987). That method uses incubation of the glycoprotein with trifluoromethanesulfonic acid at 0° C. for 0.5 to 2 hours followed by neutralization with aqueous pyridins at −20° C.
A target modification site amino acid of the glycoprotein is not accessible due to steric hindrance of glycans on one or more nearby glycosylation site amino acids. After trimming of an N-glycan or an O-glycan, a sidechain of the target modification site amino acid residue of the glycoprotein becomes accessible. Depending on the size and characteristic of the steric hindrance, the modification site amino acid residue can be adjacent to the glycosylation site amino acid, 2 residues away from the glycosylation site amino acid, 3 residues away from the glycosylation site amino acid, 4 residues away from the glycosylation site amino acid, or 5 residues away from the glycosylation site amino acid. In another example, the nearby glycosylation site amino acid can be greater than 5 residues away from the modification site amino acid but may be in proximity to the modification site due to secondary (e.g., folding), tertiary, or quaternary structure of the glycoprotein.
Depending on the size of the glycans on the glycosylation site amino acid(s), the sidechain of the modification site amino acid residue can be about 1 nm away from the steric hindrance caused by glycans attached to the glycosylation site amino acid, about 2 nm away from the steric hindrance, about 3 nm away from the steric hindrance, about 4 nm away from the steric hindrance, of about 5 nm away from the steric hindrance. In another example, the sidechain of the modification site amino acid residue is greater than 5 nm away from the steric hindrance due to reagent or catalyst size.
Attachment of a reagent to an accessible modification site amino acid residue can be done by direct attachment of the reagent or by first attaching one or more linkers to the modification site amino acid residue. Attachment of the reagent can be accomplished by enzymatic or chemical methods. Examples of protein labeling reagents are compounds with reactive groups that facilitate covalent binding with proteins. N-hydroxy succinimide esters, for example, will attach to amino groups in proteins, such as lysine residues. Maleimide reactive groups selectively react with protein residues containing a sulfhydryl group, such as cysteines. Conjugates with hydrazide reactive groups can attach via carbonyl groups. Reagents are available for the attachment of conjugates, dyes, and other moieties to proteins. These labeling reagents, which can proceed with a simple reaction and purification step, can be useful in attaching conjugates such as gold, biotin, fluorophores, and dyes to purified proteins and antibodies. Example downstream applications of labeled proteins can include immunoassays, Western blots, and immunohistochemistry.
In another example, after trimming of an N-glycan or an O-glycan, a reagent or a linker can be attached to the core glycan on the trimmed N-glycan or O-glycan. The reagent or linker can be further attached or bridged to the modification site amino acid residue. Upon hydrolysis or trimming of the glycans attached to a glycoprotein, multiple enzymes such as TGase and galactosyltransferases can be used to construct dual and multipurpose macromolecule conjugates (as illustrated in
The technology provides further derivatization of glycoproteins at two or more sites. A wide array of functionalization can be achieved by TGase-mediated bioconjugation. Various amine substrates can be attached including heterobifunctional, branched, noncanonical and proteolytically cleavable. Moreover, both endogenous and exogenous stimuli-responsive linkers can be incorporated, e.g., to create photo-responsive molecules (Moulton, et al., 2019). Since TGase bioconjugation is orthogonal to other approaches, multi-functionalization can be achieved by a combination of TGase and other chemo-enzymatic tools such as galactosyltransferases or glycosynthases (Scallon, et al., 1995; Tsai, et al., 2017; Manabe, et al., 2019). New and creative conjugates are envisioned that combine distinct modalities, e.g., protein-antibody conjugates or virus-antibody conjugates (Park, et al., 2020).
The technology can provide design of the glycoprotein entity, such as conjugation at a single or dual site(s) on an antibody. Methods and reagents to trim a glycan of a glycoprotein and their related fragments (i.e., glycan remodeling) using an endoglycosidase enzyme are provided. By remodeling the core glycan, the methods and reagents can be used to modulate effector function, e.g., ADCC or complement-dependent cytotoxicity.
The methods disclosed herein maintain core glycans in glycoproteins and in antibodies. No generation of charge variants is required. Multi-functionalized glycoproteins can be readily produced via attachment at two sites: i.e., glycan and glutamine residues. The technology can be utilized to construct antibody drug conjugates, protein drugs, PEGylated drugs.
Prior to the technology disclosed herein, it was unpredictable whether a trimmed glycan would allow a reagent or a TGase catalyzed reaction to access a modification site amino acid residue, and if so, how efficient an attempted modification would be. In an example, near quantitative conversions are observed for both steps (trimming and TGase) and for multiple antibodies. The results suggest that other glycan trimming enzymes, such as EndoF1, EndoD and EndoH, can be equally successful. Further conjugation via the remaining glycans may be used, as demonstrated by GlyCLICK (Genovis), in which saccharides can be installed onto the glycans, e.g., for glycan remodeling and/or installation of additional bioconjugation handles.
The methods can modulate Fc-related biological activity. The site-specific methods can be used to modulate the functions of glycoproteins, for example, to decrease non-specific binding of labeled antibody-based imaging agents to fragment-gamma receptors (FcγRs) on immune cells (Gao, P., et al., 2015). The resulting conjugates using this methodology are likely to have abolished or markedly reduce effector function. For imaging applications, this feature is useful by decreasing nonspecific binding such as reducing binding of the Fc domain to Fcγ receptors on immune cells.
Conversely, to enhance effector activity, unnatural glycan substrates such as oxazolines can be reintroduced, after trimming a glycan, using a glycosynthase and/or removal of fucose using fucosidases to recover the effector function. The glycan remodeling process enables fine-tuning of the glycans and thus facile modulation of Fc-related biological activity, e.g., ADCC that are mediated by Fc receptors. Both model antibodies utilized (cetuximab and Infliximab) have effector function. To completely abolish this activity, EndoS can be utilized. Conversely, to enhance this activity, unnatural glycan substrates (such as oxazolines) can be re-introduced using a glycosynthase and/or removal of fucose using fucosidases to recover the effector function.
A novel method is presented to modify antibodies that has surprising advantages. First, it does not require complete removal of Fc domain's N-glycan. Second, unlike the previous procedures, the resulting antibody does not generate a charge variant, while maintaining the primary sequence (i.e., native form of the antibody) and core glycan (GlcNAc). Overall, the products are homogenous with only a single glycoform, well-defined, efficacious and more likely to have reduced immunogenicity and greater stability than the previous methods.
Trimmed glycans on antibodies render glutamine residue(s) accessible for transamidation reaction, e.g., mediated by TGase. Transglutaminase (TGase, e.g., E.C. 2.3.2.13) is a family of enzymes that catalyzes acyl transfer reactions of the unsubstituted amide in glutamine (Gln or Q) sidechain in proteins via a thioester intermediate (see
A simple, robust and adaptable system is presented to generate photoremovable protein conjugates. The utility is validated by generation of a photoactivable protein, E. coli polymerase manager UmuD. These dynamic switches in proteins that impart spatial and temporal control are valuable to manipulate biological systems. An approach is presented to label glutamines with an 15N isotope in native peptides and proteins. This process obliviates the need for metabolic labeling and/or recombinant production, which are not readily accessible to many proteins and proteoforms. See U.S. Pat. No. 11,129,790B2, Chemo-Enzymatic Site-Specific Modification of Peptides and Proteins to Form Cleavable Conjugates.
Glycans play essential and critical roles in the structures and functions of numerous proteins. For immunoglobulins (IgG), a major function is a defense mechanism in humans against pathogens and foreign agents. For example, glycans are involved in humoral immune response through interactions between N-glycans on IgG with Fcγ receptors on immune cells to mediate effector function such as ADCC or complement-dependent cytotoxicity. Furthermore, variations in the glycosylation patterns may lead to pharmacodynamic, pharmacokinetic, and stability differences in protein pharmaceuticals, which influences the antibody's product quality, safety, and efficacy.
These methodologies can produce homogenous antibody conjugates and their related fragments for various applications such as analysis, imaging, radioimmunoconjugates, cytotoxicity using antibody-drug-conjugates (ADCs), PEGylation, and glycoengineering.
Surprisingly, all amine-containing molecules evaluated are excellent substrates for microbial transglutaminase (compounds 1 and 2;
Modifications using compounds 1 and 2 in
Since the overall yield is quantitative, it is shown that both fucosylated and non-fucosylated or glycan species are modified, which further expands the utility of the technology. The TGase-catalyzed transformation results in a modification at a single site (Gln295) in the heavy chain (HC) of the antibody (
Infliximab (Remicade) was tested and showed similar results (
The biological activity of antibody conjugates, i.e., cetuximab conjugated to Alexa Fluor647 (
Non-specific methods, which include acylation of amines (e.g., lysines and the N-terminus) or alkylation of thiols (e.g., cysteines), have been used to assemble various glycoproteins. Some glycoproteins are approved by the U.S. Food and Drug Administration such as antibody drug conjugates (ADCs) (Chari, et al., 2014; Beck, et al., 2017). Over 100 ADCs are currently in clinical trials, but specific methods of making ADCs are urgently needed.
The previous methods for modifying glycoproteins have several limitations. First, the reactions lack site-specificity and result in heterogenous mixtures. In general, kinetically controlled, these processes also suffer from poor reproducibility and are prone to side-reactions (Luo, et al., 2016). Second, modification at or near the functional domains such as the complementarity-determining regions of antibodies is likely to perturb the structure and thus function. These multiple sites of modification have been shown to affect the protein's activity (e.g., antibody) negatively, for example, reducing the binding affinity and specificity. Third and lastly, characterization of non-specific conjugation is cumbersome, as before conjugation, many proteins already have various post-translation modifications (PTMs) such as deamidation, oxidation, glycosylation, cross-linking, or other reactive metabolites. See, e.g., Liu et al., 2016; Chumsae et al., 2015; Chumsae et al., 2014; Klaene et al., 2014; Liu et al., 2014; Chumsae et al., 2013; and Dai et al., 2013. Accounting for these PTMs, the number of unmodified and modified species increases exponentially after non-specific bioconjugation. This synthetic approach makes it difficult to detect all modifications and often leads to underestimation. For example, sites of modification are missed because of the prevalence of false negatives during analysis. To overcome these limitations, the technology disclosed herein provides site-specific methods that are predictable, can be carried out with few steps and offer new possibilities for glycoprotein engineering.
The hydrolysis of N-glycans using cetuximab (Erbitux, chimeric IgG1) was examined as a model system. Both endoglycosidase (EndoS) and amidase (PNGase F) hydrolyzed N-glycans on the constant domain (Fc) of cetuximab and various antibodies (IgG1-4), albeit with several differences. First, hydrolysis or trimming by EndoS (an endoglycosidase; EndoS and EndoS2 are used interchangeably) retained the innermost glycan N-acetylglucosamine (GlcNAc), whereas hydrolysis by PNGase F removed the glycan completely. Second, hydrolysis by EndoS retained the antibody's asparagine residue, whereas hydrolysis by PNGase F (an amidase) underwent deamidation which led to conversion of asparagine residue into aspartic acid (Asp297, carboxylic acid sidechain,
A comparison of PNGase F and EndoS mediated hydrolysis of cetuximab is presented in
The isoelectric points (pI) of the EndoS-treated antibody (pI 7.8-8.2;
Subsequent site-specific TGase-mediated conjugation of polyethylene glycol (PEG) and chromophore (fluorophore) onto cetuximab is shown in
Additional trimming reactions were carried out to study transamidation. The reactions contained 18 μM cetuximab (Erbitux, Selleckchem, A2000) or 17 μM Infliximab (Remicade, European Pharmacopoeia, Y0002047) and 25 mM tris-buffered saline (TBS) pH 7.4, and was initiated with immobilized EndoS2 GlycINATOR (E.C. 3.2.1.96, Genovis; AO-GL6-010, per manufacturer instructions) at 37° C., and for 3 h and 40 min. To remove excess unreacted reagents, each reaction mixture was desalted using 30 kDa molecular weight cut-off (MWCO) centrifugal filters (Amicon unit, UFC503096) into 25 mM TBS pH 7.4, prior to the transamidation reaction.
After Endo-S mediated trimming of glycan in cetuximab, an evaluation of various amine-containing substrates using microbial transglutaminase (mTGase), which converts unsubstituted amides on glutamines into substituted amides, was conducted. This enzyme was utilized because it has been shown that mTGase has broad specificity towards the amines with two main features: primary amines are generally accepted and substituents at the alpha position significantly slows the reaction down (Gundersen, M., et al., 2014). All amine-containing molecules evaluated were excellent substrates for mTGase (e.g., compounds 1 and 2;
The transamidation reaction contained 25 mM TBS pH 7.4, 100 mM 2-azidoethanamine (compound 1,
The concentrations of the peptides and proteins were determined using UV absorption at 280 nm and extinction coefficients based on amino acid sequences. All aqueous solutions were prepared using Milli-Q water. All cell culture methods were performed with aseptic technique in a biosafety cabinet.
As illustrated in
These modifications were confirmed by attachment of 10 kDa polyethylene glycol (PEG-10 kDa, compound 4,
The SPAAC reaction contained 25 mM TBS pH 7.4, 200 μM Alexa Fluor 647® azide (AF647, compound 3,
The reaction was quantitative, as determined by a complete shift in the electrophoretic mobility of the antibody's heavy chain (HC) conjugated to a 10 kDa PEG (
Cetuximab contains complex biantennary Fc N-glycans that are highly heterogenous (both fucosylated and non-fucosylated exist) and most species are fucosylated (Qian, J., et al., 2007). Since the overall yield was quantitative, it was deduced that both fucosylated and non-fucosylated or afucosylated glycan species were modified, which further expanded the utility of the approach.
The TGase-catalyzed transformation resulted in a modification at a single site [glutamine (Gln, Q) 295] in the heavy chain (HC) of the antibody (
The loading ratio was calculated based on the ratio of the absorption of the chromophore and antibody. The absorbance spectra of antibody-chromophore conjugates were collected from 220 to 750 nm (NanoDrop spectrophotometer, ND-1000). The concentration of the chromophore AF647 or AF488 was calculated using the absorbance of the conjugates at 650 nm or 495 nm and extinction coefficient of 270,000 M−1cm−1 or 73,000 M−1cm−1 respectively, provided by Click Chemistry Tools. The antibody concentration was estimated by subtracting the chromophores contribution at 280 nm and the calculated extinction coefficient based on its amino acid sequence (217,440 M−1cm−1 for cetuximab; 203585 M−1cm−1 for Infliximab). The summary of each conjugate's ratio is provided in Table 2.
Reducing SDS-PAGE of the cetuximab-chromophore conjugates is shown in
Reducing SDS-PAGE of the cetuximab-chromophore conjugates is shown in
Nonreducing SDS-PAGE of the cetuximab-chromophore conjugates is shown in
Reducing SDS-PAGE of the cetuximab-chromophore conjugates is shown in
Nonreducing SDS-PAGE of the cetuximab-chromophore conjugates is shown in
The enzymatic reaction can be further optimized to increase the DAR by increasing the temperature, reaction time or enzyme concentration to achieve a maximum loading ratio of 2, as described in the literature (Jeger, S., et al., 2010; Dennler, P., et al., 2014). To further expand the scope, Infliximab (or Remicade) was evaluated and found that various amine-containing clickable handles and fluorophores (
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Bio-Rad Mini-PROTEAN 3 system or Criterion Cell. First, the reaction mixture was incubated with SDS Sample Buffer at 80° C. for 10 min. For reducing and nonreducing gels, 4× reducing SDS sample buffer (Boston Bio Products, BP-110R) and 2× nonreducing SDS sample buffer (Bio-Rad, 1610737) were used, respectively. Second, the samples were loaded into 12% tris-tricine precast protein gels (Bio-Rad, 4561044) or 4-15% tris-glycine precast protein gels (Biorad, 5671083). Precision Plus Protein™ Dual Xtra Prestained Protein Standards (Bio-Rad, 1610377) were used for mass calibration. Electrophoresis was then performed at 200 V for 20 min. The gel was stained by Coomassie R250 and then destained using 10% acetic acid and 40% methanol. The gels were imaged using an iBright FL1000 Imaging system (Thermo Fisher Scientific).
Reducing SDS-PAGE of infliximab-chromophore conjugates is shown in
Reducing SDS-PAGE of infliximab-chromophore conjugates is shown in
Isoelectric focusing was performed using a Bio-Rad Criterion system. First, the reaction mixture was mixed in 1:1 dilution with isoelectric focusing sample buffer (Bio-Rad, 1610763). Second, the samples were loaded into pH 3-10 Criterion isoelectric focusing precast gel and placed into the cell. Then, 1× Anode buffer (Bio-Rad, 1610761) and 1× Cathode buffer (Bio-Rad, 1610762) were placed in the upper and lower chamber of the criterion cell, respectively. Third, isoelectric focusing was run initially at 100 V for 1 h to initiate desalting of the sample, followed by higher voltage at 250 V for 1 h to mobilize the antibody, and lastly 500V for 30 min to complete electro-focusing. The gel was stained by Coomassie R250 and Crocein Scarlet and then destained using 10% acetic acid and 40% methanol. The gels were imaged using an iBright FL1000 Imaging system.
NIH:OVCAR-3 (Ovcar3) ovarian carcinoma cells (HTB-161, American Type Culture Collection, ATCC) were cultured in the recommended RPMI-1640 media (ATCC, 30-2001) supplemented with 20% heat-inactivated fetal bovine serum (R&D Systems, S11150H) and 0.01 mg/mL bovine insulin (Sigma-Aldrich, I0516) in a humidified incubator at 37° C. and 5% CO2. Media was replenished every 2-3 days and cells were passaged in T75 culture-treated flasks (Thermo Scientific, 12-565-350) at 70-90% confluency.
Ovcar3 cells were harvested and plated in a 24-well plate with #1.5 cover glass (P24-1.5H-N, Cellvis) at 30,000 cells per well in 1 mL of cell culture media. Cells were incubated for 48 h. Cet-AF647 staining solution was prepared by diluting Cet-AF647 to 5 ng/μL in cell culture media. Media from each well was aspirated and replaced with 200 μL of 5.0 ng/μL staining solution or with fresh media and incubated for 1 h at 37° C. prior to imaging. 15 min prior to imaging, 2.5 μg/mL of Hoechst 33342 (Invitrogen, H3570) was added to each well. The plate was then imaged using confocal fluorescence microscopy (Zeiss LSM800) with a 40× objective. All imaging parameters were kept consistent throughout imaging.
The biological activity of the antibody conjugates, i.e., cetuximab conjugated to Alexa Fluor 647, was assessed via confocal fluorescence microscopy in a cancer cell line (Ovcar3) that highly expresses epidermal growth factor receptor (EGFR). Under physiological conditions, the antibody conjugates bound and internalized into the cytoplasm of Ovcar3, as evident from the fluorescence signal (
This data confirmed that the conjugate's biological activity was preserved.
Ovcar3 cells were harvested and resuspended at 5×105 cells/mL in 1 mL of media with and without 0.5 ng/μL, of cetuximab-AF647 (Cet-AF647). Cells were incubated for 1 hour at 4° C. in the dark. Each sample was washed twice and resuspended in 1 mL of phosphate buffered saline (10-010-023, Gibco). Cells were analyzed via flow cytometer (Attune NxT, Thermo Fisher) equipped with 635 nm laser. Stained and unstained cells were sampled 6 times. Cells were gated and measured for mean AF647 fluorescence using Attune NxT software. Results were compiled and analyzed in Prism 8 (GraphPad Software).
All thermal stability assays were carried out using a 96-well plate with a final volume of 20 μL on a Bio-Rad CFX96 Real-Time PCR Detection System. Samples for all antibody conjugates were prepared at concentrations of 0.2 mg/mL. The assays were performed in PBS pH 7.4 with a 10× concentration of SYPRO Orange (Invitrogen). Samples were heated from 20 to 100° C. at a rate of 0.5° C./min with fluorescence measurements recorded at 0.2-degree increments. Melting temperatures were calculated from ΔF/ΔT values based on a minimum of three replicates. Standard deviation is reported with listed values in Table 3 below.
U.S. Pat. No. 11,129,790B2 is hereby incorporated by reference in its entirety.
This application claims priority to U.S. Provisional Application No. 63/187,376, filed 11 May 2021, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/028859 | 5/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63187376 | May 2021 | US |
