Covalent Chemical Ligation Strategy for Mono- and Polyclonal

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Covalent chemical ligation strategy for monoclonal and polyclonal immunoglobulins at the nucleotide binding sites Diana Lac, Chun Feng, Gaurav Bhardwaj, Huong Le, Jimmy Tran, Li Xing, Gabriel Fung, Ruiwu Liu, R. Holland Cheng, and Kit S. Lam Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00574 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015

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Bioconjugate Chemistry

Covalent chemical ligation strategy for monoclonal and polyclonal immunoglobulins at the nucleotide binding sites †

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Diana Lac , Chun Feng , Gaurav Bhardwaj , Huong Le , Jimmy Tran , Li Xing , Gabriel Fung , Ruiwu † § † #‡ Liu , Holland Cheng , Kit S. Lam , , * D.L., C.F., G.B., K.S.L. designed research. D.L., C.F., H.L., J.T., L.X., G.F. performed research. D.L., C.F., G.B., K.S.L. analyzed data. D.L. designed the figures and wrote the text. All authors read and edited the manuscript. †

#

‡

Department of Biochemistry and Molecular Medicine, Division of Hematology & Oncology, UC Davis

NCI-designated Comprehensive Cancer Center, University of California, Davis, Sacramento, California §

95816; Department of Molecular & Cellular Biology, University of California, Davis, Davis, California 95616 * To whom correspondence may be addressed. Email: [email protected]. KEYWORDS Nucleotide binding pocket, covalent crosslinker, monoclonal antibody, polyclonal antibody, antibody drug conjugate

1 ACS Paragon Plus Environment


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1 2 3 4 5 6 ABSTRACT 7 8 9 Non-specific ligation methods have been traditionally used to chemically modify immunoglobulins. Site10 11 specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the 12 13 fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took 14 advantage of the reported nucleotide-binding pocket (NBP) in F(ab) arms of immunoglobulins by 15 16 developing indole-based, 5-difluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries, for the 17 identification of affinity elements that can be used as site-specific derivatization agents against both 18 19 monoclonal and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at 20 21 the NBP. Immunoconjugates resulted from such affinity elements can be used as therapeutics against 22 cancer or infectious agents. 23 24 25 26 27 28 TOC Graphic 29 30 31 32 33 34 35 36 37Figure 2. Indole-3-butryic acid directs the peptide to the nucleotide binding pocket (NBP) where free amines reside. At 38slightly basic pH, DNFB reacts with the several free amines within this binding pocket which allows covalent ligation 39peptide to bind to the free amines located within the NBP. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 NH

NH 2

NO 2

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Indole Peptide-Peg linker- Lys(biotin)

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pH 8.5, RT, 1h

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ACS Paragon Plus Environment

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Introduction Targeted therapy using monoclonal antibodies (mAbs) has revolutionized the treatment of cancer by recognizing antigens expressed on cell surfaces (1, 2). The addition of cytotoxic drugs to these mAbs, creating antibody-drug conjugates (ADC) is a natural extension of this approach, and it has achieved varying success in the clinic. These antibody drug conjugates combine the targeted specificity of mAbs with the enhanced tumor-killing power of toxic effector molecules permit effective discrimination between target and normal tissue, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs (3-5). Recently, trastuzumab emtansine (Kadcyla; Genentech/Roche), an antiHer2 maytansine conjugate has demonstrated an improved survival compared to standard treatment (6). Similarly, brentuximab vedotin (Adcetris; Seattle Genetics) received accelerated approval for the treatment of relapsed Hodgkin lymphoma or relapse systemic anaplastic large cell lymphoma through the use of a CD30 directed antibody conjugated to a microtubule-disrupting agent monoomethyl auristatin E (MMAE) (7). The first clinically approved ADC, gemtuzumab ozogamicin (Mylotarg; Wyeth/Pfizer), however, was removed from the market because of toxicity and lack of efficacy in larger clinical trials (8). Although these approvals show great potential of ADCs to impact major unmet needs in oncology, further work is necessary to optimize this new class of therapeutic agents. Cytotoxic drugs are generally conjugated to antibodies through nonspecific methods, via (i) primary amines of lysine side chains or (ii) free sulfhydryls from cysteines by reduction of the hinge and inter-strand disulfide bonds (9). These modifications, however, creates heterogeneous products because of the prevalence of the same functional groups throughout the antibody. These complex mixtures lead to variable in vivo pharmacokinetics, efficacy, and safety profiles (2). The high abundance of lysine in each immunoglobulin molecule, for example, makes it difficult to control the stoichiometry and specificity of the chemical conjugates. Furthermore, in cysteine conjugation, the stability of the conjugation site is dependent on the structural and chemical environment surrounding the site (10). Therefore, to create a homogenous product, extensive purification processes are typically needed. Many investigators have moved towards the development of site-specific modifications to create ADCs, providing complete control over the attachment thereby decreasing heterogeneity in the final clinical products. Current site-specific conjugation methods include both antibody engineering and chemical methods. Several research groups have engineered a cysteine residue on the surface of the antibody to introduce sulfhydryl groups for subsequent conjugation to a linker payload (2, 11). Schultz et al (12, 13) has incorporated unnatural amino acid p-acetylphenylalanine to site specifically conjugate the payload



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through a stable, non-cleavable oxime linkage. These methods have successfully created homogenous conjugates. In addition, a number of chemical methods have emerged by modifying N-terminal residues (14, 15), targeting tyrosine residues (16, 17), or adding recognition tags for enzymatic modification (1820). While these approaches can site-specifically conjugate a payload to antibodies, many of these methods require the use of organic solvents which are often too harsh to maintain folded protein structures (21) or require very extensive conjugation times which may affect protein stability. Conjugation to the N-terminus, for example, can also be problematic as the conjugation of a cytotoxic payload or a ligand may affect epitope binding which is typically located at the N-terminus. A simple alternative strategy for site-specific derivatization of clinically available antibodies is to develop novel site-specific elements that bind to and then covalently ligate to unique site on the surface of immunoglobulin molecules through proximity ligation. Here we have exploited the use of a ligand that is known to bind directly to the Fab arms of the immunoglobulin molecule. This approach is economically attractive because it can be applied to many existing clinically available antibodies, avoiding additional steps of protein bioengineering, reducing production costs and shortening preclinical-to-clinical translation times (22). Rajagopalan et al (23) have identified a nucleotide-binding pocket (NBP), which exists in all immunoglobulin Fab arms. This highly conserved pocket is located between the variable light (VL) and variable heavy (VH) domain of all antibody isotypes (23-25). Through an in silico docking study, Handlogten et al (25) identified indole-3-butryic acid as a highly specific compound that binds to the NBP with Kds ranging between 1μM to 8μM, with binding affinity dependent on the antibody (23, 25). Alves et al (26) has described a UV photo-crosslinking method relying on the indole group to crosslink to specific residues within the NBP of IgG. This method requires the use of UV excitation to develop an indole radical used for crosslinking. This requirement for UV excitation increases the risk of impairing Fc recognition and creates a loss in the antibody’s ability to recognize its antigen. Together, there is an increased need to discover chemoreactive affinity elements that can take advantage of this NBP for site specific ligation. Unlike other site-specific ligation techniques that link cytotoxic payload to a specific amino acid on the immunoglobulin, here we describe site-specific ligation as specificity to the NBP of the Fab domain. We have exploited the site specificity provided by indole-3-butryic acid and developed focused one-bead-one-compound (OBOC) combinatorial peptide libraries capped by indole-3-butyric acid to discover an affinity element capable of site-specific ligation to the nucleotide-binding site of immunoglobulin via proximity-ligation. For irreversible ligation to free amines and thiols at the nucleotide-binding site, we placed 1,5-difluoro-2,4-dinitrobenzene (DFDNB) (27) adjacent to the indole group of the OBOC library. This crosslinking agent, in conjunction with our targeting ligand, creates a


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superior conjugate because of the mild conditions required for covalent ligation to the NBP of the antibody. From this library we have discovered several affinity elements that allows site-specific introduction of orthogonal functional groups that can be readily used for subsequent conjugation of cytotoxic payload to generate ADC. This linker can also be used for the ligation of toxins such as MMAE to make antibody-toxin conjugates for cancer therapy. This can be done through the use of existing clinically available mAbs such as Herceptin, Avastin, and Rituxan. In addition, one may site-specifically introduce disease-specific targeting ligands onto the readily available polyclonal human intravenous immunogobulins (IVIGs) without the need to reengineer an antibody, which takes months to years to develop. This latter application is particularly useful for rapid deployment of neutralizing antibodies against emerging pathogens in epidemics and pandemics, such as the recent MERS-coronavirus and Ebola virus outbreaks. Results and Discussion The increasing interest in antibody drug conjugates and bispecific antibodies presents great need for site-specific linkers to control conjugation to antibodies. Here, we present a small molecule peptidic affinity element that has the ability to covalently conjugate to the NBP located in the Fab domains of monoclonal and polyclonal antibodies. This simple conjugation strategy allows for the addition of pathogen or cell targeting ligand or a toxic payload to be easily adapted for the development of antibody drug conjugates. Design and Screening of a Peptide Library The design of a site-specific conjugation peptide or affinity element is based on targeting the NBP in IgG antibodies. In our studies, trastuzumab was used as the model monoclonal antibody. Based upon previous molecular modeling data (25) and the published Fab domain crystal structure of trastuzumab (28), we located the NBP with H101, H103 and L36 as the amino acids interacting with indole-3-butyric acid. In this NBP, numerous lysine residues (at least 3) were found at the vicinity of the indole-3-butryate binding site (Figure 1a). Based upon this structural characteristic, a small virtual library of compounds was examined (Supplementary Figure 1) by comparing the docking conformations and energies to the NBP. Taking advantage of the lysine residues located at the vicinity of NBP, we expected to have enhanced affinity of indole-3-butryate to the NBP by including negatively charged amino acids in the library (Supplementary Table 1). The energy characterizations obtained from Autodock (29) indicated that placing the lysine residue with crosslinker displayed at the side chain [Lys(crosslinker)] directly next to indole-3-butryic acid was preferred over other conformations (Supplementary Table 1). Compounds with amino acids between the indole-3-butryate and Lys (crosslinker) had less optimal mean cluster



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binding energies.

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Results of this in silico screen predicted that indole-3-butryate-Lys(acryloyl)-Glu

would bind with the highest affinity to the NBP. Using this knowledge, we designed eight OBOC libraries to screen for compounds with enhanced affinity and crosslinking ability to the NBP.

(A)

(B)

(C) NH NH 2

NO 2 O 2N F

NO 2

Indole Peptide-Peg linker- Lys(biotin)

N H

HN O

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pH 8.5, RT, 1h Indole O Peptide-Peg linker- Lys(biotin)

Figure 1. (A) Depicted here is one of the F(ab) arms used in the Autodock studies. Indole-3-butyric acid (in pink) can be seen nestled within the nucleotide binding pocket. Selected residues are color-coded: lysine (blue), cysteines (yellow), and arginine (green). (B) Based upon the top clusters generated from Autodock, several libraries were designed. X1X2X3X4 library with DFDNB is shown. (C) Indole-3-butryic acid directs the peptide to the nucleotide binding pocket (NBP) where a few lysine residues with primary amines reside. At slightly basic pH, FDNB reacts with one of these lysines, resulting in covalent ligation of the affinity element to the NBP.

OBOC combinatorial libraries based on the results of the in silico screens were synthesized on TentaGel® beads using a split-mix technique (30). KX1, KX1X2, KX1X2X3, and KX1X2X3X4 libraries Ncapped by “indole-butyrate-Lys(crosslinker)” were synthesized with the combination of unnatural and Lamino acids (Figure 1b, Supplementary Figure 2, Supplementary Table 2). The variable portions of the libraries were synthesized using the split-mix technique. The N-terminal Lys residue was then N-capped with indole-3-butyric acid, and its side chain reacted with 1,5-difluoro-2,4-dinitrobenzene (DFDNB) to generate 1-amino-5-fluoro-2,4-dinitrobenzene (FDNB). The reactivity of the remaining fluorine is significantly lower than that of the first two unreacted fluorine of DFDNB (27). Covalent ligation of FDNB to a primary amine of the target protein can be achieved by simply raising the pH of the buffer slightly to 8.5, making the reaction very mild, efficient, convenient, and easy to control (Figure 1c). We have also prepared similar OBOC libraries but use acrylic acid or cyanuric chloride, instead of DFDNB,


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to cap the Lys side chain. However, for some unknown reasons, these resulted in very high background, which made screening difficult (data not shown). In order to identify beads that covalently link to trastuzumab, we have modified our previously reported enzyme-linked colorimetric screening method (31). After incubating trastuzumab with the FDNB-derivatized bead library at pH 8, we treated the beads with acidic glycine (pH 3.0) buffer to ensure that only covalently linked trastuzumab will remain on the bead. The covalently linked antibody was then detected with anti-human antibody-alkaline phosphatase conjugate followed by the BCIP substrate. Positive peptide-beads turned turquoise and were physically isolated for microsequencing. All of the FDNB-libraries were screened multiple times, ensuring that ~90% of all possible permutations were screened. Although our computational analysis concluded that negatively charged amino acids was the best fit for NBP, we included all natural amino acids in our library construction as some of them may be required for optimal binding (Supplementary Table 2). Furthermore, we want to ensure that true positive leads that differed from virtual prediction would not be missed. The result of the library screening is shown in Figure 2a. Several trends were observed from the positive compounds. Asp and Phe (including unnatural amino acid derivatives) appeared frequently in all forms of the library. In the peptides without Asp, other negatively charged amino acids were found. This is consistent with the notion that multiple Lys residues in the vicinity of NBP favor the identification of negatively charged amino acids. The positive sequences were resynthesized on TentaGel beads to compare their ability to covalently capture trastuzumab (Figure 2a,b). Similar incubation times and washes were performed as in the screening process. After the acidic glycine wash step, anti-human IgG conjugated to Cy3 was used to detect the crosslinked trastuzumab. From this study, several indole-butyrate-Lys(FDNB)-derivatized dipeptides with Asp, Glu, and Phe(2Cl)-Thr-Phe(2Cl)-Gln were found to have superior crosslinking abilities compared to other hits and our negative control (indole-K(FDNB) without peptide). These results were consistent with the results found in the computer-modeling data. In order to understand if the indole-butyrate-peptides were merely a strong binding agent to the NBP or if FDNB was utilized to covalently crosslink the Lys residues in the NBP, we first treated the indole-butyrate-peptide-beads with methylamine or 4-methylpiperidine to block the remaining unreacted fluorine of FDNB. As expected, we found that using similar crosslinking incubation times and staining procedures as the reconfirmation assay, Cy3 intensity of the indole-butyrate-Lys(FDNB)-Asp-Ser linker (affinity element DS) beads decreased significantly after treatment with these highly basic compounds.



Such treatment decreased the fluorescent intensity indicating that the ligation of the peptide compound to the NBP of the immunoglobulin was greatly impaired (Figure 2c). (A)

(B)

Indole Linker Ile Leu Ser Asp His Phe Glu Asn Asp-Ser Phe-Bpa Asp-Leu Asp-Gly Trp-Glu Bpa-Gln Asp-Thr Trp-Phe-Gln Phe-Asp-His Asp-Trp-Nva Glu-Asp-Pro Trp-Phe(2Cl)-Thr-Thr Thr-HoCit-His-His Phe(2Cl)-Thr-Phe(2Cl)-Gln Glu-Leu-Gln-Nal2 Glu-HoPhe-Gln-His Glu-Ala-Asn-Glu Asp-Gly-Phe(2Cl)-Thr

trastuzumab

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Figure 2. (A) All of the hits identified in the library screen were resynthesized on TentaGel. Trastuzumab was added to the beads, washed, and covalent ligation was promoted by increasing the pH to pH 8.5. Non-ligated antibodies were washed away with acidic glycine buffer (pH 3.0). Anti-human Cy3 IgG was added to the beads to probe the covalently attached trastuzumab. Beads were then imaged and quantified using Image J. Average intensity of beads were taken. Different color indicates the libraries that the hits were from. X1 (blue), X1X2 (red), X1X2X3 (green), and X1X2X3X4 (purple). (B) Describes the overall concept of the bead staining. (C) The FDNB on Asp-Ser beads were reacted with methylamine or methylpypridine prior to staining with antibodies. Such pretreatment resulted in a 50% decrease in fluorescent intensity. * P < 0.05.

Peptide Crosslinker Characterization To understand the site specificity of our peptide to the NBP, the top two hits (affinity element DS, compound 1, and affinity element F(2,3-diCl)-T-F(2,3-Cl)-Q, compound 2) from the reconfirmation studies were resynthesized in soluble biotinylated form (Figure 3a). Here, the term affinity element includes indole-K(FDNB)-peptide-PEG linker-K(biotin). An indoylyl control affinity element (compound 3) without the amino acid peptide sequence was also synthesized. PEG linkers were added between the indole-peptide and biotin to ensure that avidin can bind to the biotinylated immunoglobulin without any steric hindrance. The antibody peptide-linker reaction chemistry is shown in Figure 1c.


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Reducing, denaturing conditions were employed for Western blot analysis to confirm the covalent reactions and to obtain distinguishable bands between unreacted IgG and the biotinylated IgG (Figure 3b). After one-hour incubation of 50μM of biotinylated affinity elements with 10μM trastuzumab at pH 7.5, the pH was raised to 8.5 to initiate covalent ligation. The resulting biotin-linker-antibody conjugates were detected at 25kDa and 50kDa for all 3 affinity elements, representing covalent ligation to both the heavy and light chains (Figure 3b). This is expected as residues from both chains form the NBP. Controls (antibody alone and peptide alone) did not show any bands at any molecular weight. For unknown reason, the promising hit affinity element F(2,3-diCl)-T-F(2,3-Cl)-Q (compound 2) showed similar results as our indolyl control affinity element (compound 3) (Figure 3b), where no differences were observed among the different pH. The faint bands at pH 7.5 indicate some reactivity of FDNB at neutral pH. (A)

(B) O 2N

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Figure 3. (A) Structure of soluble affinity element DS (1), Phe(2Cl)-T-Phe(2Cl)-Q (2, F(2Cl)), and indolyl control (3) used in solution phase experiments. (B) Western blot analysis of biotinylated trastuzumab (DS - Lane 1 and 2, F(2Cl) – Lane 5 and 6, indoylyl control (Ind) – Lane 7 and 8). When pH was increased to 8.5, we observed an increase in product for the DS sample. Some product is still seen at pH 7.5 due to peptide specificity to the nucleotide binding pocket and mild reactivity at pH 7.5. No differences, however, were seen with Phe(2Cl). Same amount of product was observed in both pH 7.5 and 8.5. Free peptide and free trastuzumab, as predicted, was negatively stained.

Affinity element DS (compound 1), however, showed a dramatic increase of product at pH 8.5. This is encouraging, since indole-3-butyrate is known to have some specificity in the NBP (32) but the addition of Lys(FDNB), Asp and Ser dramatically increased the amount of peptides capable of binding to and covalently modify this pocket. Alves (33),(34) recently reported the use of an indole-3-butyric acid based



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linker as a photoaffinity label to directly modified the NBP with UV irradiation. Ten to twenty fold excess of photoaffinity label was used in their experiment. We repeated similar experiment with a related photoaffinity label but had to use 50 fold excess of reagent in order to achieve significant antibody derivatization (Supplementary Figure 3).

In contrast, only 2.5 fold excess of affinity element DS is

required for antibody derivatization.

Site Specificity Characterization Classical peptide mapping techniques were first used to determine the linker conjugation site using trypsin and chymotrypsin for protein digestion of the separated heavy and light chain, followed by analysis with mass spectrometry. Unlike other site-specific conjugation linkers, which modified one specific amino acid (35, 36), our peptide linker has the ability to ligate to one of the several Lys residues in the vicinity of the NBP, making the analysis more difficult. Furthermore, the two aromatic nitro-groups present in the affinity elements greatly suppress ionization, making mass spectrometry analysis even more difficult. As a result, we were unable to obtain a conclusive result using this approach. Our next approach was to perform partial enzyme digest followed by western blot analysis to determine site-specific ligation. We first biotinylated each of the four immunoglobulins (trastuzumab, IVIG, rituximab, and cetuximab) with the affinity element DS (compound 1), digested them with papain and then treated the samples with 2-mercaptoethanol to generate reduced Fab and Fc fragments, which could be resolved on SDS-PAGE. Due to the glycosylation of the Fc fragments, reduced Fc is slightly bigger than reduced Fab (25KDa). The result of this study is shown in Figure 4. In all antibody conjugates, only the reduced Fab fragment (Figure 4a), but not the reduced Fc portion was biotinylated, as reflected by the single positive band in western blot. This is expected since NBP resides in the Fab region and the affinity element DS is expected to ligate to this fragment (32). In a control experiment sulfoNHS-biotin, a nonspecific amine reactive reagent (Figure 4c), was used to derivatize the same set of antibodies, both Fab and Fc fragments were labeled as expected (Figure 4b). Together, this data proves that our ligation strategy is general, site-specific, ligate to the Fab portion of the immunoglobulin only, and can be applied even to polyclonal IVIG. The fact that affinity element DS can site-specifically ligate to the Fab in many clinical antibodies without the need to reengineering the protein is a significant improvement to current site-specific ligation technology for antibodies.


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For morphologic confirmation of site-specific ligation, we mixed the antibody conjugate with streptavidin-nanogold and then analyzed the final immunoconjugate with cryo-electron microscopy (Supplementary Figure 4). We were able to visualize nanogold (1.4 nm) binding to only the inner arms of IgG, at positions adjacent to both Fab arms (Supplementary Figure 4a,b). It appeared that there were two residing sites for nanogold on each Fa One of the nanogolds is localized to the variable domain, which is the known location of the NBP. The other nanogold was located near the constant domain. The distance between two resident sites is about 6nm, which is in good agreement with the size of streptavidin (5.6nm). Since the EM image is a two-dimensional projection, this distance suggests the flexibility of the peptide and the multiple conjugation sites on streptavidin, as well as the difference of the binding orientation in respect to Fab arm. The second conjugation site, which appeared to be at the constant domain, can be bound to the NBP but its location in the EM image is a result of the flexibility of the peptide and streptavidin conjugation. It should be noted that no binding was seen in the Fc arm or on the outer portion of the Fab further demonstrating consistency of site-specific conjugation. These two sites



were also observed when F(ab)2 fragments were imaged. The nanogold was again found at positions close to the variable domain (Supplementary Figure 4c,d). In all cases, the nanogold is at a distance equivalent to one streptavidin from the Fab arm again confirming the site specificity provided by the indole peptide. These imaging studies were repeated and similar results were obtained (data not shown). Our antibody-linker conjugate (TrastDS) generated a single 150kD band (Figure 5a) on a nonreducing SDS-PAGE gel indicating no loss of interchain disulfide bonds during the conjugation reaction. With the abundance of primary amines on IgG, FDNB can potentially randomly react with any of the primary amines on the surface of the antibody. The absence of additional products indicates that conjugation did not occur randomly but was site-specific. (B) TrastDS

(A)

trastuzumab

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trastuzumab (µM) 10 DS (µM) 50 pH 7.5

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trastuzumab (µM) 10 10 10 10 10 DS (µM) 10 25 50 75 100 pH ---------------7.5---------------

10 10 10 10 10 10 25 50 75 100 ----------------8.5--------------

50kDa

25kDa Reduced

Figure 5. (A) Western blot analysis of antibody peptide conjugate under nonreducing conditions. One band was found at 150kDa indicating a homogenous product. (B) Differences in conjugation was seen when pH was increased, with most products identified at pH 8.5. (C) Western blot analysis of soluble DS peptide. At increasing molar ratio, increased product was observed at both pH7.5 and pH8.5. At ratios above 2.5:1 peptide: trastuzumab, no additional products were produced.

The reactivity of affinity element DS was further investigated to explore optimal conditions for the ligation reaction. The optimal pH of the reaction was studied, and the result is shown in Figure 5b. After 1h the bands had increasing intensity as the pH was increased from 7.5 to 8.5 with the most products seen in 8.5. Beyond pH 8.5, no additional products were found (Supplementary Figure 5a). We


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further analyzed the optimal time for conjugation. Within 15 minutes of increased pH there is no difference in the amount of product created (Supplementary Figure 5b). In order to understand the efficacy of our affinity element, we used different ratios of affinity element DS to trastuzumab to discover the most efficient combination (Figure 5c). It was observed that at molar ratios above 2.5 affinity element DS: 1 trastuzumab, no additional products were seen on the western blot analysis. This saturability of ligation reaction further proves the site-specificity of the targeting moiety affinity element combination. Through these optimization steps, it was found that at a 2.5:1 molar ratio of affinity element DS to antibody is sufficient for efficient ligation at pH 8.5 in 15 minutes. We further analyzed our antibody conjugate by quantifying the number of biotinylated affinity element conjugated to each antibody, using a biotin quantification assay (Table 1). Free avidin was mixed with 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to form a yellow complex with molar extinction coefficient of 34000 M-1cm-1 at 500 nm. However, biotin has much higher binding affinity towards avidin than that of HABA. When biotin conjugates are mixed with the HABA-avidin complex, biotin replaces HABA to give a biotin-avidin complex that barely absorbs light at 500 nm. By measuring the absorbance difference at 500nm using UV-Vis spectroscopy, the number of avidin-accessible biotin can be calculated. Using this method, we estimated that ~2 affinity element DS were attached to each antibody, which is consistent with the two known NBPs on each IgG molecule. This number of conjugates per antibody is similar to other known site-specific antibodies (2), (5), (37), (38). As expected, the nonspecific sulfo-NHS-biotin conjugate can randomly react with many different lysine side chains on the surface of the antibodies tested, resulting in 4-9 biotin per antibody molecule. Table 1. Number of biotin per antibody molecule estimated by HABA analysis TrastDS TrastNHS Trast avidin-HRP Bevacizumab 2.30 4.47 0 1.40 Cetuximab 2.77 7.98 0 1.40 Rituximab 2.10 6.58 0 1.40 IVIG 2.21 9.09 0 1.40 Trastuzumab 1.60 6.33 0 1.40 Table 1. This table indicates the number of the biotin per monoclonal antibody. Using a HABA-avidin complex with our conjugated biotinylated antibodies, the difference in absorbance at 500nm is proportional to the number of biotin per antibody. Our DS peptide retains the site specificity that has an average biotin per antibody ratio between 1 and 3, whereas the positive control, NH2 reactive biotin to monoclonal antibodies, has an average of 5 to 8 biotin molecules conjugates to each antibodies.



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In vitro Characterization The site-specific ligation method reported here has broad utility.

For example, cytotoxic

compounds or toxins can be easily conjugated to the C-terminus of the affinity element DS, directly or through one or more ethylene glycol linker moieties, to form stable ADCs. Similar ligation sites can also be used to prepare bispecific antibodies, using the complete immunoglobulins or Fab fragments. Although the NBP is not adjacent to the antigen-binding site of immunoglobulins, it is close enough that we must demonstrate the ligation strategy does not impair the antigen binding function of the antibody. Here, we use flow cytometry to assess the binding properties of trastuzumab that had been derivatized with the affinity element DS (TrastDS) to SKBR3, MCF7, and MDA-MB-468 breast cancer cells. SKBR3 cells have high expression level of Her2 receptors, MCF7 cells have moderate Her2 expression, while MDAMB-468 cells do not have Her2 expression (Supplementary Figure 6). We used Cy3 labeled anti-human IgG secondary antibody in the flow cytometry studies (Figure 6a-c); no fluorescent shift between the (A)

(B)

(C)

Control (D)

Trast

TrastDS (F)

(E)

Control Trast bioTrastDS:Avidin:bioMMAE (1:1:3) bioTrastDS:Avidin:bioMMAE (2:1:2) bioTrastDS:Avidin:bioMMAE (3:1:1) Figure 6. (A-C) Cells (A: SKBR3; B:MCF7; C:MDA-MB-468) were incubated with 40ng/ml of Trast or TrastDS. 40ng/ml trastuzumab or TrastDS were applied to cells prior to secondary antibody labeling, Cy3-labeled anti-human IgG Fc secondary antibody. The fluorescent intensity of the secondary antibody was recorded through FACS on each cell lines. The binding of TrastDS to the NBP did not affect binding affinity of trastusumab to SKBR3 or MCF7 cells. As expected, no binding of trastusumab or trast-DS was seen in MDA-MB-468 cells. (D-E) Similar FACS analysis was performed on TrastDS-MMAE conjugates. In SKBR and MCF7 cells, the immunotoxin conjugates did not affect binding affinity of trastuzumab to the Her2(+) cells. No binding of trastuzumab or trastDS-MMAE was seen in Her2(-) cells, MDA-MB-468 cells.


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derivatized

and

underivatized

trastuzumab

was

observed.

BioTrastDS:Avidin:bioMMAE,

an

immunotoxin generated through our conjugation method retained the antigen binding ability (Figure 6df). Both Her2 expressing cell lines showed similar binding affinity and specificity to the unmodified antibody. In addition to flow cytometry, we prepared TrastDS-biotin/MMAE-biotin/neutravidin complex at two ratios (1:1:3 and 2:1:2) and evaluate their cytotoxic effects on Her2-expressing breast cancer cells (MCF7 cells and SKBR3 cells) and Her2 negative cells (MDA-MB-458). MMAE is linked to biotin via a valine-citrulline linker, which can be cleaved by cathepsin after endocytosed inside the cells (39). We therefore anticipate that (i) trastuzumab decorated MMAE complex will be much more active than MMAE complex without trastuzumab in Her2-expressing cell lines, and (ii) Her2-non-expressing cell lines will be relatively resistant to any of these conjugates. These were confirmed by the experiment shown in Figure 7. It should be noted, however, MDA-MB-468 did display some low levels of (A)

(B)

(C)

bioTrastDS bioTrastDS:Avidin:bioMMAE (1:1:3) bioTrastDS:Avidin:bioMMAE (2:1:2) bioMMAE:Avidin

(D)

MCF7

Her2 Expression *

bioTrastDS:avidin: bioTrastDS:avidin: bioMMAE (1:1:3) (µM) bioMMAE (2:1:2) (µM) 0.38 0.02

bioMMAE:avidin (4:1) (µM) 0.08

TrastDS (µM) >10

SKBr3

***

1.40

0.01

0.18

>10

MB-468

negative

>10

>10

NA

>10

Figure 7. Cells (A: SKBR3; B:MCF7; C:MDA-MB-468) were incubated with TrastDS conjugated with MMAE at either 1:3 or 2:2 ratio. In cell lines with HER2 expression, SKBR3 and MCF7, a decreased in cell viability was seen in antibodies with MMAE conjugation. In the cell line lacking HER2 expression, MDA-MB-468 this effect was not as pronounced. (D) Table indicates the IC50 values for the different avidin, TrastDS, and MMAE conjugates.

nonspecific cytotoxicity with these conjugates, which can be explained by the limited premature cleavage of MMAE from the conjugate and/or baseline non-specific uptake of the conjugate by the cells. Interestingly, conjugate with 2:1:2 ratio is significantly more potent than the conjugate with 1:1:3 ratio. This can in part be explained by the tetravalent Her-2 binding sites of the 2:1:2 conjugate versus bivalent Her-2 binding sites of the 1:1:3 conjugate, making the former conjugate with higher binding affinity and higher endocytic uptake. Additionally, it is conceivably that because biotinylated MMAE is considerably smaller than TrastDS-biotin and with less steric hindrance, more biotin-MMAE will be complexed when avidin becomes limiting, making some of the conjugates without any TrastDS-biotin. In addition to



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ADCs, the ligation chemistry described here can also be applied for the preparation of radioactive ligand conjugates or immunomodulating conjugates.

Conclusion With the clinical success of antibody drug conjugates (trastuzumab emtansine and brentuximab vedotin), there is increasing interest in the development of robust strategies for site-specific ligation of therapeutic payload to monoclonal antibodies producing homogeneous products. Heterogeneity in conjugation to antibodies could result in toxicity and antibody instability (2). We exploited previous knowledge on NBP reported by Rajagopalan (23) and Alves (33) by employing OBOC combinatorial chemistry and novel screening strategy to develop NBP affinity elements that can site-specifically ligate therapeutic pay-load to the two NBPs on an antibody molecule. Through proximity ligation, the affinity element can be covalently linked to the immunoglobulin via the built-in dinitrofluorobenzene, under very mild conditions. This conjugation results in a homogenous product that does not affect the thiol stability of the antibody. The indole-Lys(FDNB)-Asp-Ser affinity element can efficiently derivatize trastuzumab, rituximab, cetuximab and IVIG. This indicates the broad applicability of the affinity DS to cover a broad spectrum of immunoglobulins. These affinity elements can be readily adapted to other antibodies through additional OBOC library screening. Therapeutic payloads can be introduced to the immunoglobulins via two general approaches: (i) direct site-specific ligation of affinity element-therapeutic payload conjugate, or (ii) site-specific ligation of azide-derivatized affinity element, followed by introduction of alkynederivatized therapeutic payload via click chemistry (16). Compared to other site-specific covalent modifications, this technique is mild, highly efficient, and can be used for many clinically available monoclonal antibodies. This technique is simple and should be readily scalable, thereby shortening optimization time and can be quickly adapted for manufacturing antibody drug conjugates. The most unique feature of this antibody conjugation strategy is that it can be applied to polyclonal antibodies such as clinical grade IVIG, making it possible to introduce pathogenspecific ligands to IVIG, generating neutralizing antibodies against infectious microbes, such as the MERS coronavirus and the Ebola virus.


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Experimental Section Library design and synthesis To aid in the design of the library, computer-modeling studies were done. We started by performing molecular docking studies using Autodock v4.2 (29) to understand the binding of indole-3-butyric acid to trastuzumab. The three-dimensional structure of human trastuzumab available in PDB (ID: 1N8Z) was used for all docking studies. From the low energy binding conformations calculated by the indole-trastuzumab docking, the spatial and charge properties were identified. This knowledge was used to design several virtual test ligands of varying lengths and charge properties. These ligands were docked with trastuzumab using Autodock v4.2 (29). Trastuzumab and ligand structures were prepared for docking using Autodock Tools (29) package. Partial atomic charges were assigned to the ligands using the Gasteiger-Marsili method, and after merging of non-polar hydrogens; rotatable bonds were assigned using Autodock Tools. Water molecules were removed from the trastuzumab structure; the missing hydrogen atoms and Kollman partial charges were added. Further, non-polar hydrogen atoms were merged to their corresponding carbons. A grid size of 60 x 60 x 60 with grid spacing of 0.375Å for smaller ligands was used, and for larger ligands the grid size was increased proportionally to fit the whole ligand molecule. We used the Lamarckian Genetic Algorithm (Pseudo Solis-Wets Algorithm(40)) to perform 256 independent docking runs with default parameters in Autodock. Cluster analysis was performed on docked results using RMS tolerance of 2Å. The results were analyzed to compare the lowest energy binding energy conformations. Further, to test for the binding specificity profile, blind docking runs were performed using these test ligands, and trastuzumab as the target protein. SwissDock webservice (http://swissdock.vital-it.ch) (41, 42) was used to conduct these blind dockings. Energy optimized structure of the ligands was calculated using Merck Molecular Force Field (MMFF)(43) as implemented in Marvin Suite v 5.11 (http://www.chemaxon.com/products/marvin). Among the clusters generated by SwissDock, top clusters were analyzed and compared to check for convergence on the binding site. These top clusters were then used to design several one-bead-one-compound (OBOC) combinatorial libraries to identify an optimal crosslinking compound to the NBP. Library screen/confirmation screen Approximately 100,000 library beads were immobilized onto 30mm polystyrene dishes by using a series of 90% DMF washes. Beads were then washed and swelled in PBS. Nonspecific binding was inhibited with blocking buffer (0.1% BSA, 0.1% Tween, and 0.05% sodium azide) for one hour in room temperature. After several PBS washes, 1ng/ml trastuzumab (Genentech, South San Francisco, CA) in PBS (pH 7.0) was added and was placed in a rotating incubator



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at 37°C for 1 hour. Here, we used trastuzumab as the model monoclonal antibody. Excess antibody was removed, and beads were gently washed with PBS. To facilitate crosslinking of FDNB to the antibody of the peptide library, we raised the pH to 8.5 for one hour. After crosslinking, beads were washed sequentially with PBS, 100mM pH 3 glycine, and then pH 8 TBS. Anti-human IgG-alkaline phosphatase conjugate was then added at 1:1000 dilution. After washing, beads were then developed using BCIP (5bromo-4-chloro-3’-indolyphosphate p-toluidine salt) solution. (NBT (nitro-blue tetrazolium chloride) was not used due to high background staining with TentaGel library beads.) Using a dissecting microscope, plates were imaged and positive beads were isolated. The isolated beads were then treated with 8M guanidine/HCl to remove all non-covalently linked proteins. The beads were further washed with PBS and water. Beads were decoded using Edman degradation chemistry (ABI Procise 494 Protein Sequencer). In the confirmation studies, hits were synthesized on TentaGel. Staining steps were the same except 1:30 anti-human IgG conjugated to Cy3 was used to allow for fluorescence quantification. Beads were imaged with Olympus IX2-UCB (Center Valley, PA) under 4x objective. Cy3 excitation and emission settings were used. Bead intensity was measured and quantified using Image J. Western blot 50µM DS peptide was incubated with 10µM trastuzumab at 37ºC for 1hr in a shaking incubator. Samples were then dialyzed to remove unbound peptides, with frequent water changes. Crosslinking occurred by increasing the sample to pH 8.5 and incubated for 1hr at room temperature. 50ng of each sample was loaded onto 10% SDS-PAGE gel with Laemelli loading buffer containing βmercaptoethanol. Denatured, reduced samples were run @140V. Proteins were then transferred to PVDF membrane at 100V. Membranes were washed with TBST and were blocked with blocking buffer for 1hr in room temperature. After a series of PBS washes, 1:500 streptavidin-alkaline phosphatase was added to the membrane and incubated for 1hr. Blots were developed using BCIP/NBT (Promega, Madison, WI). Similar western blots were done using bevacizumab (Genentech/Roche, South San Francisco, CA), rituximab (Biogen IDEC, Cambridge, MA), cetuximab (Bristol-Myers Squibb, New York, NY), and intravenous immunoglobulin (BDI Pharma, Columbia, SC). Papain Digestion: The biotinlyated affinity element DS was conjugated with several FDA-approval therapeutic antibodies, including trastuzumab, IVIG, rituximab, and cetuximab. The affinity element and the antibody were incubated at 37°C for an hour followed by room temperature incubation with 1.4μL 7N ammonia hydroxide for 1 hour. These biotinylated immunoconjugates were then digested with papain to generate Fab and Fc fragments. Fragments were then separated on a 4% to 12% Tris-glycine gel (Life Technologies, Inc., Gaithersburg, MD) and transferred to PVDF membranes. Blots were probed with


Page 19 of 23

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streptavidin-Horseradish Peroxidase (HRP) (BioRad, Hercules, CA), which would have a strong binding affinity with biotinlyated affinity element. Blotting was carried out in 10% non-fat milk solution. Fragments

were

visualized

using

enhanced

chemiluminescence

reagents

(GE

health

Care,

Buckinghamshire, UK). Commassie Brilliant Blue G-250 (Thermo, UK) staining were performed at the same time on a separated gel with the same samples. HABA- Avidin Quantification Biotinylated antibody conjugates were prepared as described in previous sections. The absorbance of HABA/Avidin premix solution (Pierce) was measured at 500nm. The biotinylated proteins were mixed with HABA/Avidin premix solution for 30 minutes at room temperature. Absorbance measurements were measured again at the same wavelength. Calculations of moles of biotin per mole of protein used the following formula: A λ = ε λ bC, (Beer’s Law) where A is the absorbance of the sample at a particular wavelength (λ). The wavelength for the HABA assay is 500nm. ε is the absorptivity or extinction coefficient at the wavelength (λ). For HABA/avidin samples at 500nm, B is the cell path length of the microplate reader (Molecular Devices, Sunnyvale, CA) expressed in centimeters. C is the concentration of the sample expressed in molarity. Moles of biotin per mole of protein is calculated by: mmol biotin from the sample / mmol protein in original sample. Flow Cytometry 10 x 105 cells (SKBR-3, MCF7, and MDA-MB-468) were plated. Trastuzumab-peptide crosslinker was prepared as previously mentioned. Trastuzumab-peptide conjugate and/or biotinylated MMAE were then conjugated to avidin at 1:1:3, 2:1:2, and 3:1:1 molar ratio. Cells were dosed with 40ng/ml modified or naked antibodies for 2 hours at 37°C. Cells were trypsinized, washed with PBS, and incubated with anti-human IgG Fc Cy3 secondary antibody (Thermo, Waltham, MA) for 1hr on ice in the dark. Cells were then with cold PBS and resuspended for flow cytometry analysis using the FACScan (Becton Dickinson, San Jose, CA). SKBR-3, MCF7 and MDA-MB-468 cells (100,000 cells per sample) were incubated at 37°C with 40ng/ml conventional or DS conjugated trastuzumab for 2hrs in 2 ml total volume. After this incubation, cells were washed and then incubated with anti-human IgG Fc Cy3 secondary antibody (1hr on ice in dark). PBS + 1% FBS + 2 mM EDTA were used as dilution buffer for secondary antibody. Cells were then washed and analyzed by flow cytometry (Becton Dickinson, San Jose, CA). Cytotoxic Assay SKBR-3, MCF7 and MDA-MB-468 cells were plated in 96-well plates at 10,000 cells per well in triplicates, and allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. Medium was then removed and replaced by fresh culture medium containing different concentrations of



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trastuzumab or trast-DS-biotin/MMAE-biotin/neutravidin complex at two ratios (1:3:1 and 2:2:1) for 72 hours. Cell viability was detected by MTS assays (Promega, Madison, WI). Supporting Information Detailed synthesis and analytical data of molecules, experimental methods, supplementary data are provided.

Funding Sources DL acknowledges support from the Howard Hughes Medical Institute’s Med Into Grad program at UC Davis (#56006769) and Biomolecular Technology T32 fellowships (T32-GM008799). GB acknowledges support from DOD Prostate Cancer Research Program Postdoctoral Training Award PC121738.


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