One-Step Conjugation Method for Site-Specific AntibodyâDrug

Subscriber access provided by UNIV LAVAL

Article

One-step conjugation method for site-specific antibody-drug conjugates through reactive cysteine-engineered antibodies Daisuke Shinmi, Eri Taguchi, Junko Iwano, Tsuyoshi Yamaguchi, Kazuhiro Masuda, Junichi Enokizono, and Yasuhisa Shiraishi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00133 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

One-step conjugation method for site-specific antibody-drug conjugates through reactive cysteine-engineered antibodies

Daisuke Shinmi,† Eri Taguchi,† Junko Iwano,† Tsuyoshi Yamaguchi,‡ Kazuhiro Masuda,§ Junichi Enokizono,† and Yasuhisa Shiraishi *,#

†

Research Core Function Laboratories, Research Functions Unit, R&D Division, Kyowa Hakko

Kirin Co., Ltd., Tokyo, Japan. ‡

Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin

Co., Ltd., Tokyo, Japan. §

Innovative Technology Laboratories, Research Functions Unit, R&D Division, Kyowa Hakko

Kirin Co., Ltd., Tokyo, Japan. #

R&D Planning Department, R&D Division, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan.

* Corresponding Author: Yasuhisa Shiraishi, Ph. D. R&D Planning Department, R&D Division, Kyowa Hakko Kirin Co., Ltd. 1-6-1, Ohtemachi, Chiyoda-ku, Tokyo, 100-8185, Japan. 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Phone: +81-3-3282-0985 Fax: +81-3-3282-0087 E-mail: [email protected]

2 ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. Combination with direct conjugation by use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC. 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Antibody-drug conjugates (ADCs) are a new class of highly potent drug modalities that equip antibodies with potent cytotoxic payloads and have been attracting growing interest in cancer therapy in past few years. Targeted delivery of a cytotoxic drug to the target cells by antibody leads to potential reduction in its toxicity toward normal cells, thereby minimizing harmful effects. ADCs have allowed significant progress in the oncology field as demonstrated by the two FDA approved-drugs, ado-trastuzumab emtansine (KADCYLA®) and brentuximab vedotin (ADCENTRIS®). Furthermore, ADCs with various combinations of antibody and linker-drugs are currently undergoing clinical studies.1 Conjugation of the drug payload to the antibody requires a controlled chemical reaction with specific amino acid residues exposed on the surface of the antibody. Most of the current ADC products generally go through a chemical reaction of lysine ε-amino groups or cysteine sulfhydryl groups activated by reducing the inter-chain disulfide bonds.2 These conjugation chemistries produce heterogeneous products at the molecular level, each with different pharmacokinetics, efficacies, and therapeutic indexes. ADCs with an average of two to four drug-to-antibody ratios (DARs) were shown to be particularly suitable for exerting maximal in vivo efficacy.3 Site-specific conjugation is one of the most effective techniques to reduce heterogeneity of current ADC.4 In recent years, the site-specific modification of antibodies has been achieved through protein engineering strategies such as 4 ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


introducing cysteine residues in the antibody sequences,5-9 incorporating sugars,10,

11

adding

unnatural amino acid with bio-orthogonal reactivity,12, 13 or genetic encoding of peptide tags for further enzymatic modifications.14, 15 The antibody engineering strategies using cysteine residues such as THIOMAB technology,7,

16

offer a particularly convenient method, in that no

cell-engineering and additional reagents are required. However, in Chinese hamster ovary (CHO) cells, the engineered cysteine residues of IgG generally formed mixed disulfides with cysteine or glutathione, possibly during the fermentation process. Consequently, relatively complex reduction-oxidation steps are required in order to produce unpaired, reactive sulfhydryl groups prior to drug conjugation (Scheme 1A).7, 16 We previously reported that several engineered cysteine residues at less exposed positions on the Fab fragment showed high conjugation efficiency with molecules of various sizes. Among them, Fab-Lc-Q124C exhibited the highest thiol reactivity even though an engineered cysteine residue was introduced at the position with the lowest Accessible Surface Area (ASA).17 In order to confirm the versatility and potency of Lc-Q124C, IgG-Lc-Q124C was produced through fermentation in CHO cells and compared with other variants such as IgG-Hc-A118C and IgG-Lc-V205C using THIOMAB technology.16 Here, we disclosed that IgG-Lc-Q124C (named active thiol antibody: Actibody) retained high thiol reactivity itself and allowed performing a one-step conjugation without any pretreatment (Scheme 1B). Furthermore, we examined its 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

potential to be applied towards site-specific ADC.


Page 6 of 38

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. General strategy for synthesis of site-specific antibody-drug conjugates with common cysteine-engineered antibody technology (A). Simple strategy for synthesis of site-specific antibody-drug conjugates with Actibody technology (B).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

RESULTS Comparison of thiol reactivity of cysteine positional variants. Trastuzumab (Tra) was used as a model antibody in this study. Yields of all of Tra-IgG cysteine positional variants were similar to that of Tra-WT and existed mainly as monomer (Figure S1). We examined thiol reactivity and drug-antibody ratio (DAR) of four selected clones (Tra-IgG-WT, -Lc-Q124C, -Lc-V205C, and -Hc-A118C) without any reduction procedure, using 4-PDS assay and Alexa488 conjugates (Table 1). Tra-IgG-Lc-V205C and -Hc-A118C are well-known for production of site-specific antibody-drug conjugates with THIOMAB technology.7,

16

As with the previous case of

Tra-Fab,17 selected cysteine positional variants existed as monomer and possessed similar binding affinity to that of Tra-IgG-WT (data not shown). Tra-IgG-WT showed less thiol reactivity and Alexa488-antibody ratio (0.074 and 0.17, respectively). Tra-IgG-Lc-V205C and -Hc-A118C exhibited low thiol reactivity (0.48 and 0.36, respectively), which was far from the theoretical value: 2.0. In addition, Alexa488-antibody ratios were also low (0.44 and 0.37, respectively), which was consistent with low thiol reactivity. In contrast, Tra-IgG-Lc-Q124C retained much higher thiol reactivity and Alexa488-antibody ratio, which were similar to the theoretical values. In order to examine the differences in thiol reactivity and Alexa488-antibody ratios, we determined the molecular weight of selected clones by LC/MS analysis (Figure 1). Tra-IgG-Lc-V205C and -Hc-A118C showed broaden peaks that were located at higher molecular 8 ACS Paragon Plus Environment

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


weight than those previously reported.7 On the contrary, Tra-IgG-Lc-Q124C showed a single peak corresponding to the estimated value and so was the case for Tra-IgG-WT.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 38

Table 1. Thiol reactivity and Alexa488-antibody ratios of selected clones.

a

Variant

Thiol Reactivitya

Alexa488a

Tra-IgG-WT

0.074 ± 0.0022

0.17 ± 0.0087

Tra-IgG-Lc-Q124C

2.0 ± 0.016

2.0 ± 0.013

Tra-IgG-Hc-A118

0.36 ± 0.0025

0.37 ± 0.060

Tra-IgG-Lc-V205C

0.48 ± 0.0040

0.44 ±0.0057

Thiol reactivity and Alexa488-antibody ratios were determined by three independent assays. The

mean value and standard deviation (SD) were computed from 3 independent measurements.


Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Deconvoluted mass spectra of naked Tra-IgG-WT (A), Tra-IgG-Lc-Q124C (B), Tra-IgG-Hc-A118C (C), and Tra-IgG-Lc-V205C (D). The most intense peak in each mass spectrum corresponds to the molecular weight of the antibody.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

Preparation and evaluation of Tra-IgG-vcMMAE conjugates. Three-quarters of the ADCs undergoing current clinical trials utilize either auristatin or maytansine derivatives, which are developed by Seattle Genetics and ImmunoGen.18 Auristatin derivative, vcMMAE, has been designed to bind to antibodies via maleimide-thiol chemistry. Considering the high thiol reactivity and DAR of Alexa488 conjugates, Tra-IgG-Lc-Q124C was directly conjugated with vcMMAE to produce site-specific ADC (Scheme 1B). Tra-IgG-Lc-V205C-vcMMAE and -Hc-A118C-vcMMAE were prepared as controls through the reduction/oxidization procedure shown in a previous report (Scheme 1A).19 As shown in Figure S2, all of the Tra-IgG-vcMMAE conjugates

mainly

stood

as

a

population

with

DAR:

2.0.

We

confirmed

that

Tra-IgG-Lc-Q124C-vcMMAE existed mainly as monomers by SEC analysis (Figure S3). Hence, we confirmed that Tra-IgG-Lc-Q124C could produce site-specific conjugates using the one-step procedure shown in Scheme 1B. In order to compare the ADC derived from Tra-IgG-Lc-Q124C with those derived from Tra-IgG-Lc-V205C and -Hc-A118C, we assessed cytotoxicity against Her2 expressing SK-BR-3, BT-474,

and

NCI-N87

cells

using

CellTiter-Glo

assay.17

We

confirmed

that

control-IgG-Lc-Q124C-vcMMAE had no cytotoxicity between 0.32 and 1000 ng/mL (Figure S4). Tra-IgG-Lc-Q124C-vcMMAE

exerted

similar

cytotoxicity

to

those

of

Tra-IgG-Hc-A118C-vcMMAE and –Lc-V205C-vcMMAE against SK-BR-3 cells in vitro (Figure 12 ACS Paragon Plus Environment

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2A). Similar results were observed in BT-474 and NCI-N87 cells (Figure 2B, C). IC50 values of Tra-IgG-vcMMAE conjugates were shown in Table S1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

Figure 2. In vitro cytotoxicity of Tra-IgG-Lc-Q124C-vcMMAE, Tra-IgG-Hc-A118-vcMMAE, Tra-IgG-Lc-V205-vcMMAE, and control-IgG1-Lc-Q124C-vcMMAE on Her2-positive breast cancer cell lines, SK-BR-3 (A), BT-474 (B), and NCI-N87 (C).


Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Analysis of drug-linker stability in vitro. To confirm that the stability depended on the conjugation

site,

Tra-IgG-Lc-Q124C-Alexa488,

-Lc-V205C-Alexa488,

and

-Hc-A118C-Alexa488 were incubated in human plasma for 96 hours followed by size-exclusion chromatographic analysis through detection of Alexa488 fluorescence. As reported by Shen’s group,16 the fluorescent peak of Tra-IgG-Lc-V205C-Alexa488 was in great part retained throughout a 24 hours incubation period, whereas that of Tra-IgG-Hc-A118C-Alexa488 slightly decreased with a new peak appearing at lower molecular weight after 24 hours, which corresponded to the albumin-Alexa488 conjugate (Figure S5A and B). On the contrary, the fluorescent intensity of Tra-IgG-Lc-Q124C-Alexa488 was dramatically moved to the lower molecular weight peak at 24 hours (Figure S5C). The time course of the ratio of Tra-IgG-Alexa488 conjugate was plotted (Figure 3), which also exhibited dramatic reduction caused by retro-Michael Reaction in Tra-IgG-Lc-Q124C-Alexa488 conjugate.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

Figure 3. Stability of Tra-IgG-Lc-Q124C-Alexa488, Tra-IgG-Hc-A118-Alexa488, and Tra-IgG-Lc-V205-Alexa488 in human plasma. Measurement of Tra-IgG-Alexa488 conjugate concentration was conducted by size exclusion chromatography with fluorescence detection.


Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Linker stabilization on anion exchange resin and evaluation of stabilized ADC. Recent studies demonstrated that retro-Michael type mediated transfer of linker-payload portion was induced by exposure of high concentration of free thiols such as cysteine, glutathione, and serum albumin.20 Hydrolysis of succinimide ring in alkaline medium allowed preventing retro and reversible maleimide transfer.21-23 In this study, we applied anion exchange chromatography techniques to induce succinimide ring opening under alkaline conditions in a solid phase system (Scheme 2). Succinimide hydrolysis of Tra-IgG-Lc-Q124C-Alexa488 was confirmed using LC/MS analysis (Figure S6). The Tra-IgG-Lc-Q124C-vcMMAE hydrolyzed conjugate (hy-Tra-IgG-Lc-Q124C-vcMMAE) was produced following the same procedure. The binding and cytotoxic activities of hy-Tra-IgG-Lc-Q124C-vcMMAE were maintained (Table S2, Figure S7). The hy-Tra-IgG-Lc-Q124C-Alexa488 retained the linker-drug portion in human plasma during the 96 hours incubation period (Figure 4).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

Scheme 2. Mechanism of drug transfer from an antibody to albumin through the maleimide exchange reaction and prevention of drug transfer by succinimide hydrolysis with anion exchange chromatography techniques.


Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Evaluation of succinimide hydrolyzed Tra-IgG-Lc-Q124C-Alexa488. Stability of hy-Tra-IgG-Lc-Q124C

conjugate,

non-treated

Tra-IgG-Lc-Q124C

Tra-IgG-Lc-V205C conjugate in human plasma.


conjugate

and


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

Antitumor activity of Tra-IgG-Lc-Q124C-vcMMAE. In Drake’s and Jackson’s reports,14, 24 a NCI-N87 tumor xenograft model was used for evaluation of efficacy of Trastuzumab-based ADC. To evaluate whether differences in linker stability in vitro correlate with differences in antitumor activity

in

vivo,

Tra-IgG-Lc-Q124C-vcMMAE,

hy-Tra-IgG-Lc-Q124C-vcMMAE,

and

Tra-IgG-Lc-V205C-vcMMAE were intravenously administered at once into tumor-bearing mice at doses of 0.3 and 1 mg/kg (Figure 5). All Tra-IgG-vcMMAE conjugates showed partial inhibition

in

tumor

growth

at

0.3

mg/kg.

The

tumor

growth

rates

of

both

hy-Tra-IgG-Lc-Q124C-vcMMAE and Tra-IgG-Lc-V205C-vcMMAE treated mice were slower than that of Tra-IgG-Lc-Q124C-vcMMAE treated individuals. At a dosing of 1 mg/kg, there were no differences in efficacy between these three treatment groups until day 63. However, tumor volume in mice treated with Tra-IgG-Lc-Q124C-vcMMAE started to grow again, being about three-fold

larger

than

those

of

hy-Tra-IgG-Lc-Q124C-vcMMAE

Tra-IgG-Lc-V205C-vcMMAE-treated mice at day 83.


and

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. In vivo efficacy of Tra-IgG-Lc-Q124C-vcMMAE, hy-Tra-IgG-Lc-Q124C-vcMMAE and Tra-IgG-Lc-V205-vcMMAE in a xenograft model. Male SCID mice were injected subcutaneously

with

NCI-N87

cells.

Tra-IgG-Lc-Q124C-vcMMAE,

hy-Tra-IgG-Lc-Q124C-vcMMAE and Tra-IgG-Lc-V205-vcMMAE were dosed intravenously at 0.3 and 1 mg/kg on day 0.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

DISCUSSION Various types of site-specific conjugation technology have been reported, which mainly consist of recombinant4, 7, 10-15 and non-recombinant methods.25, 26 Engineered cysteine technology was a convenient method for which cell engineering or special additives were not required. On the contrary, a reduction-oxidation process was needed to produce free reactive cysteines. Alternative non-recombinant methods have been developed for construction of homogeneous ADC, in which bi-functional linkers such as bis-sulfone linker and bifunctional dibromomaleimide are used for the reaction with two adjacent cysteine thiol groups on the antibody.25, 26 However, these methods also need full reduction process prior to conjugation that can potentially lead to misfolding events and yield several conjugate isoforms. Regarding methods with no pretreatment involved, utilization of unnatural amino acid12, 13 was superior to other methods, however, it needs cell engineering and expensive unnatural amino acid additives. Considering these characteristics, engineered cysteine technology without oxidization would provide an ideal way for site-specific conjugation. Hc-A118C and Lc-V205C variants showed high thiol reactivity when produced in E. coli,6 while their thiol reactivity was almost lost with mammalian cells.7 Based on the observation that Tra-IgG-Hc-A118C IgG and -Lc-V205C exhibited broad peaks in LC-MS analysis, free cysteine and glutathione could be conjugated with engineered cysteines during incubation, thereby 22 ACS Paragon Plus Environment

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


capping thiol groups of the engineered cysteine.7 Surprisingly, the results presented here demonstrate that the engineered cysteine residues in Tra-IgG-Lc-Q124C exist as reduced form and exhibit higher thiol reactivity, which defies the common assumption that cysteine mutants are easily capped. Serum albumin is one of the known native proteins with a single unpaired cysteine residue as Lc-Q124C.27 Cys34 of human serum albumin (HSA) in blood is mainly present in reduced form (75%) under an aerobic condition.28 Intriguingly, ASA value of Cys34 (0.7%) is very low, which is similar to Lc-Q124C (1.1%).17 The crystal structure of HSA27 suggest that the sulfur atom of Cys34 is protected by the side chains of surrounding amino acids. On the other hand, ASA values of A118C (39.9%) and V205C (30.6%), which are calculated by the previous procedure17 are much higher than that of Lc-Q124C. Based on these facts, location at much buried position or orientation of side-chain would contribute to protecting the engineered cysteine from oxidization. In order to understand the detail mechanism of Lc-Q124C, further study such as structural analysis could be needed. Site-specific Lc-Q124C-IgG conjugates (MMAE and Alexa488) are produced by direct conjugation without any pretreatment. Tra-IgG-Lc-Q124C-vcMMAE exhibited in vitro cytotoxicity

similar

to

that

of

Tra-IgG-Lc-V205C-vcMMAE

conjugate

through

reduction-oxidization process, suggesting that IgG-Lc-Q124C could be applied to site-specific ADC. IgG-Lc-Q124C variant showed high thiol reactivity in the case of transient and stable 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

expression in CHO cells, furthermore we confirmed high thiol reactivity also in the case of preliminary fed-batch fermentation procedure (approx. 1 g/L). From our previous report,17 Fab-Lc-Q124C showed the highest thiol reactivity among selected engineered cysteine mutants, and could produce 1:1 conjugates with functional molecules of various sizes such as MMAE and branched PEG (20 kDa x 2). In this regard, both Fab and IgG of Lc-Q124C could be used for site-specific conjugation with desired molecules by simple one-step procedure. Thiosuccinimide linkage, which was formed by the reaction of maleimide electrophiles with thiol of engineered cysteine residues, causes the maleimide exchange reaction with Cys34 in serum albumin, leading to the linker’s instability in blood and consequent low efficacy in vivo.20 A recent study revealed that the basic environment surrounding conjugation sites, such as Lc-V205C, undergoes spontaneous succinimide hydrolysis, resulting in resistance to maleimide exchange reaction.16 In fact, Tra-IgG-Lc-V205C-Alexa488 was stable in human plasma, while Tra-IgG-Lc-Q124C-Alexa488 and -Hc-A118C-Alexa488 were decomposed over time. Tumey et al22 demonstrated that the reaction at pH 9.2 and 37°C naturally facilitated succinimide hydrolysis; however, this aqueous method only yields about 50% of hydrolyzed product in the case of maleimidocaproyl linker. In order to enhance the stability of Lc-Q124C-conjugates, we created a simple and efficient method based on this approach, in which the reaction is performed on anion exchange resin at pH 9.5. As a result, the hydrolyzed Lc-Q124C (hy-Lc-Q124C) 24 ACS Paragon Plus Environment

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conjugate was produced after short time exposure (6 hours), and buffer exchange was easily completed using an elution procedure. We found that hy-Tra-IgG-Lc-Q124C-Alexa488 ensured higher stability than original Tra-IgG-Lc-Q124C-Alexa488, and no differences in stability were detected between hy-Tra-IgG-Lc-Q124C-Alexa488

and

Tra-IgG-Lc-V205C-Alexa488.

hy-Tra-IgG-Lc-Q124C-vcMMAE

exhibited

similar

efficacy

than

Additionally, that

of

Tra-IgG-Lc-V205C-vcMMAE. Altogether, these results suggest that the instability of maleimide-thiol linker of Lc-Q124C-conjugate could be overcome by alkaline hydrolysis or substitution

with

more

stable

linkers.

Bromoacetamidecaproyl29

and

methylsulfonylbenzothiazole30, 31 linkers were developed as alternative agents for stable thioether conjugation. In addition, it was demonstrated that the thiosuccinimide linkage was automatically hydrolyzed, even under a neutral condition, by the incorporation of a basic functional group adjacent to the maleimide.21,

23

Recently, new palladium reagents were developed for thiol

specific conjugation of biomolecules.32 A combination of Lc-Q124C variants and these linker technologies could provide a stable antibody-drug conjugate. In conclusion, Lc-Q124C (Actibody) was prepared in a reduced form in the case of Fab in E. coli and IgG in mammalian cell line, which was contrary to the assumption that engineered cysteine is easily oxidized. Actibody is easily prepared through a substitution with cysteine at 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

Q124 (Kabat numbering) in light chains of aimed antibody and incubation. It has high capacity for payload size. These findings, put altogether, suggest that Actibody based on our new concept for introducing cysteine residues with low ASA values provides an easy method for preparing and analyzing site-specific conjugates, and shed light on the progress made regarding the analysis of antibody-conjugated functional molecules.


Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL PROCEDURES Materials.

Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl

auristatin E (vcMMAE) was obtained from MedChem Express (Princeton, NJ). Alexa Fluor 488 C5 maleimide was purchased from Thermo Fisher Scientific (Waltham, MA). Anti Tra-IgG clones 16712 and 18018 were purchased from AbD Serotec (Oxford, UK). HRP Antibody Conjugation Kit was also purchased from AbD Serotec. Chinese hamster ovary (CHO)-K1 cell line was obtained from European Collection of Cell Cultures and cultured at 37°C with 5% CO2 in EXCELL325PF (SAFC Biosciences, Lenexa, KS) supplemented with 4 mM L-glutamine and 50 µg/mL gentamicin. The reaction buffer composed of 20 mM citrate (pH 6.0) and 150 mM NaCl. The composition of conjugation buffer was 50 mM Tris-HCl (pH 7.5) and 5 mM EDTA. All cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). Human breast cancer cell line: SK-BR-3 was cultured at 37°C with 5% CO2 in McCoy's 5A supplemented with 10% fetal bovine serum. Human breast cancer cell line: BT-474 was cultured at 37°C with 5% CO2 in RPMI1640 supplemented with 10% fetal bovine serum and 10 µg/ml bovine insulin. Human gastric cancer cell line: NCI-N87 was cultured at 37°C with 5% CO2 in RPMI1640 supplemented with 10% fetal bovine serum.

Animals. 6-7 week-old male C.B17/Icr-scid Jcl (SCID) mice were purchased from Clea (Tokyo, 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

Japan). All animal studies were performed in accordance with Standards for Proper Conduct of Animal Experiments at Kyowa Hakko Kirin Co., Ltd. under the approval of the company’s Institutional Animal Care and Use Committee (protocol number APS15J0135 and APS15J0233). Fuji Research Park and Tokyo Research Park of Kyowa Hakko Kirin co., Ltd. are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International.

Expression and purification of cysteine-engineered Tra-IgG. The light or heavy chain genes of Tra-IgG, which contained cysteine mutations, were cloned into a mammalian expression vector. Cysteine positions in Cκ light chain (Lc) and CH1 heavy chain (Hc) constant regions were defined by Kabat number and EU number, respectively. Each cysteine-engineered Tra-IgG (Tra-IgG-Lc-Q124C, –Lc-V205C, and -Hc-A118C) was stably expressed in CHO-K1, and these transfectants were incubated for about 1 week. The culture supernatant was applied onto 1 mL MabSelect SuRe Protein A resin (GE Healthcare, Piscataway, NJ). The resin was washed with 20 mL of D-PBS. The bound antibody was eluted with 5 mL of 100 mM glycine-HCl (pH 3.5) and neutralized using 1 M Tris-HCl (pH 8.0). The buffer exchange into reaction buffer was accomplished using Amicon Ultra-4 device (Merck Millipore, Billerica, MA). The thiol reactivity of each cysteine-engineered Tra-IgG was measured by 4-PDS method as previouly,17 except that 28 ACS Paragon Plus Environment

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 µM of Tra-IgG variant was used. Theoretically, thiol reactivity of a molecule with two free thiols is 2.0. SEC analysis and binding analysis by Biacore were carried out as described previously. 17

Preparation of Tra-IgG-Alexa 488 and vcMMAE conjugates. Tra-IgG-Lc-Q124C, -Lc-V205C, and –Hc-A118C were adjusted to 50 µM with reaction buffer and conjugated with 10-fold molar excess of Alexa Fluor 488 C5 maleimide at 4°C overnight. The excess Alexa Fluor 488 C5 maleimide was purified and buffer-exchanged with reaction buffer using Amicon Ultra-4 device. The concentrations of antibody Alexa488 conjugates were calculated according to the manufacturer's instructions. Tra-IgG-Lc-Q124C was adjusted to 30 µM with reaction buffer and conjugated with 10-fold molar excess of vcMMAE in the presence of 20% v/v acetonitrile solution at 4°C overnight. The excess vcMMAE was purified and buffer-exchanged with D-PBS using Amicon Ultra-4 device. The yield of Tra-IgG-Lc-Q124C was approx. 80%. Preparation procedure has room for improvement. Tra-IgG Lc-V205C and –Hc-A118C needed to have their introduced cysteine residues reduced before conjugation. Tra-IgG-Lc-V205C and –Hc-A118C were adjusted to 5 mg/mL with conjugation buffer and reacted with 40-fold molar excess of dithiothreitol (DTT) to reduce the cysteine residues at room temperature for 16 hours. The excess DTT was removed and buffer-exchanged with reaction buffer using Amicon Ultra-4 device. To 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 38

re-oxidize the interchain disulfide bonds, the reduced antibodies were adjusted to 50 µM and reacted with 15-fold molar excess of (L)-Dehydroascorbic acid (DHAA) at room temperature for 3 hours. Re-oxidized antibodies were conjugated with 2.5-fold molar excess of vcMMAE in the presence of a 20% v/v acetonitrile solution at room temperature for 1 hour. The excess vcMMAE was purified and buffer-exchanged as described above. Formation of MMAE conjugate was confirmed by LC-MS analysis. The concentrations of Tra-IgG-MMAE conjugates were determined by UV-VIS analysis and equations that have been described previously by Hamblett et al.3

Mass spectrometric analysis. All of the Tra-IgG-vcMMAE conjugates were treated with 5 units of PNGase F (Roche Diagnostics, Germany) at 4°C overnight. LC/MS analysis was performed as described previously. Briefly, samples were separated by isocratic high performance liquid chromatography (HPLC) on ACQUITY UPLC BEH200 SEC column (Waters Corporation, Milford, MA) and identified by positive time-of-flight SYNAPT high-definition mass spectrometry (Waters Corporation). Data collection and processing were controlled by MassLynx 4.1 software.

In vitro cytotoxicity. In vitro cytotoxicity analyses were performed as described previously. 30 ACS Paragon Plus Environment

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Briefly, SK-BR-3 (3,000 cells/well), BT-474 and NCI-N87 (10,000 cells/well) were seeded in clear bottom, white wall 96-well plates (Greiner Bio-One, Kremsmünster, Austria). After 24 hours, serial dilutions of trastuzumab vcMMAE conjugates were added to the cells and incubated at 37°C for 120 hours. Cell viability was then determined using the CellTiter-Glo luminescent assay (Promega Corp, Madison, WI) following the manufacturer's instructions. 17

In vitro plasma stability. Before sample preparation, human plasma was centrifuged at 20,400 × g for 10 minutes followed by filtering through a 0.22-µm filter unit (Merck Millipore). Tra-IgG-Alexa488 conjugates and -vcMMAE conjugates were diluted at 100 µg/mL with human plasma and incubated at 37°C for 0 hour, 24 hours, 48 hours, 72 hours, and 96 hours. After incubation, each sample was kept frozen until analysis. The 0-hour samples were immediately frozen after the conjugate dilution step. Tra-IgG-Alexa488 and albumin-Alexa488 conjugates were detected by fluorescence-detection size exclusion chromatography using Prominence HPLC system (SHIMADZU, Kyoto, Japan). Approximately, 90 pmols were injected into the TSKgel G3000SWXL columns (TOSOH, Tokyo, Japan). The mobile phase was the reaction buffer. The flow rate was 1 mL/min for 20 min. The percent mean stability was calculated by dividing the peak area of Tra-IgG-Alexa488 conjugate by Tra-IgG-Alexa488 and albumin-Alexa488 conjugates. 31 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

Stabilization of Tra-IgG-Lc-Q124C conjugate. Stabilization of Tra-IgG-Lc-Q124C conjugates was conducted on an anion exchange resin. Tra-IgG-Lc-Q124C conjugates were diluted 5 times with 20 mM Bis-tris propane (pH 9.5). Each diluted sample was injected onto 1-mL HiTrap Q column (GE Healthcare) equilibrated in 20 mM Bis-tris propane (pH 9.5). Columns were left at room temperature for 6 hours. Stabilized Tra-IgG-Lc-Q124C conjugates were then eluted with 20 mM Bis-tris propane (pH 9.5) and, 1 M NaCl and then buffer-exchanged with D-PBS using Amicon Ultra-4 device (Merck Millipore). The yield of stabilized Tra-IgG-Lc-Q124C conjugate was approx. 90%.

In vivo efficacy. In vivo efficacy of Tra-IgG-vcMMAE conjugates was evaluated in a NCI-N87 human gastric carcinoma xenograft model. SCID mice were injected intraperitoneally with anti-asialo GM1 (300 µg/mice, Wako Pure Chemical), then NCI-N87 cells were subcutaneously inoculated into right flank of mice at 5 x 106 cells per mice. Seven days after implantation, mice bearing palpable tumor were assigned into groups so that average tumor volume/group would be equivalent. Saline and Tra-IgG-vcMMAE conjugates were intravenously administered into mice on day 0 at dosages of 0.3, 1, and 3 mg/kg. Tumor masses were measured by calipers twice weekly, and tumor volume was calculated as tumor volume (mm3) = (length x width x width) / 2.


Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENTS The authors thank Eri Suzuki-Takanami and Yoshiko Ishikawa-Matsumoto for their technical assistance with

the

LC-MS

instrument,

Emiko

Honma for establishing the each

cysteine-engineered antibody expression vector and stably expressing cells, Aiko Uchida for performing ADC physical stability studies, and Etsuko Koshimura for performing all in vivo efficacy studies. We would like to thank Editage (www.editage.jp) for English language editing.

Funding Sources We have no funding to report in this manuscript.

Supporting Information IC50 values, binding kinetics, deconvoluted mass spectra, and SEC data of parepared IgG and conjugates.This material is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS ADC, antibody drug conjugate; ASA, accessible surface area; DAR, drug-to-antibody ratio; Her2, human epidermal growth factor receptor 2; IgG, immunoglobulin G; Tra, trastuzumab; Lc, light chian; Hc, heavy chain; used codes (e.g. Lc-Q124C), cysteine variant at 124 in Lc; 4-PDS, 33 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4,4'-dithiodipyridine; SEC, Size exclusion chromatography; and KD, dissociation constant.


Page 34 of 38

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1)

Mullard, A. (2013) Maturing antibody-drug conjugate pipeline hits 30. Nat. Rev. Drug.

(2)

Discov. 12, 329-32. Chari, R. V., Miller, M. L., and Widdison, W. C. (2014) Antibody-drug conjugates: an

(3)

emerging concept in cancer therapy. Angew. Chem. Int. Ed. Engl. 53, 3796-827. Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M., Lenox, J., Cerveny, C. G., Kissler, K. M., Bernhardt, S. X., Kopcha, A. K., Zabinski, R. F., et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063-70.

(4)

(5) (6)

(7)

(8)

(9)

(10)

(11)

(12)

Agarwal, P., and Bertozzi, C. R. (2015) Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 26, 176-92. Voynov, V., Chennamsetty, N., Kayser, V., Wallny, H. J., Helk, B., and Trout, B. L. (2010) Design and application of antibody cysteine variants. Bioconjugate Chem. 21, 385-92. Junutula, J. R., Bhakta, S., Raab, H., Ervin, K. E., Eigenbrot, C., Vandlen, R., Scheller, R. H., and Lowman, H. B. (2008) Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J. Immunol. Methods 332, 41-52. Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., Chen, Y., Simpson, M., Tsai, S. P., Dennis, M. S., et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925-32. Stimmel, J. B., Merrill, B. M., Kuyper, L. F., Moxham, C. P., Hutchins, J. T., Fling, M. E., and Kull, F. C., Jr. (2000) Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies. J. Biol. Chem. 275, 30445-50. Lyons, A., King, D. J., Owens, R. J., Yarranton, G. T., Millican, A., Whittle, N. R., and Adair, J. R. (1990) Site-specific attachment to recombinant antibodies via introduced surface cysteine residues. Protein Eng. 3, 703-8. Zhou, Q., Stefano, J. E., Manning, C., Kyazike, J., Chen, B., Gianolio, D. A., Park, A., Busch, M., Bird, J., Zheng, X., et al. (2014) Site-specific antibody-drug conjugation through glycoengineering. Bioconjugate Chem. 25, 510-20. Li, X., Fang, T., and Boons, G. J. (2014) Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew. Chem. Int. Ed. Engl. 53, 7179-82. Zimmerman, E. S., Heibeck, T. H., Gill, A., Li, X., Murray, C. J., Madlansacay, M. R., Tran, C., Uter, N. T., Yin, G., Rivers, P. J., Yam, A. Y., et al. (2014) Production of 35 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

site-specific antibody-drug conjugates using optimized non-natural amino acids in a (13)

cell-free expression system. Bioconjugate Chem. 25, 351-61. Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., Phuong, T., Barnett, R., Hehli, B., Song, F., et al. (2014) A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. U. S. A. 111, 1766-71.

(14)

Drake, P. M., Albers, A. E., Baker, J., Banas, S., Barfield, R. M., Bhat, A. S., de Hart, G. W., Garofalo, A. W., Holder, P., Jones, L. C., et al. (2014) Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjugate Chem. 25, 1331-41.

(15)

(16)

(17)

(18) (19)

(20) (21) (22)

(23)

Dennler, P., Chiotellis, A., Fischer, E., Bregeon, D., Belmant, C., Gauthier, L., Lhospice, F., Romagne, F., and Schibli, R. (2014) Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjugate Chem. 25, 569-78. Shen, B. Q., Xu, K., Liu, L., Raab, H., Bhakta, S., Kenrick, M., Parsons-Reponte, K. L., Tien, J., Yu, S. F., Mai, E., et al. (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30, 184-9. Shiraishi, Y., Muramoto, T., Nagatomo, K., Shinmi, D., Honma, E., Masuda, K., and Yamasaki, M. (2015) Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation. Bioconjugate Chem. 26, 1032-40. Hamilton, G. S. (2015) Antibody-drug conjugates for cancer therapy: The technological and regulatory challenges of developing drug-biologic hybrids. Biologicals 43, 318-32. Bhakta, S., Raab, H., and Junutula, J. R. (2013) Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers. Methods in Molecular Biology. Volume 1045: Antibody-Drug Conjugtes (Ducry, L., Ed.) pp 189–204, Springer, New York. Baldwin, A. D., and Kiick, K. L. (2011) Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjugate Chem. 22, 1946-53. Fontaine, S. D., Reid, R., Robinson, L., Ashley, G. W., and Santi, D. V. (2015) Long-term stabilization of maleimide-thiol conjugates. Bioconjugate Chem. 26, 145-52. Tumey, L. N., Charati, M., He, T., Sousa, E., Ma, D., Han, X., Clark, T., Casavant, J., Loganzo, F., Barletta, F., et al. (2014) Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy. Bioconjugate Chem. 25, 1871-80. Lyon, R. P., Setter, J. R., Bovee, T. D., Doronina, S. O., Hunter, J. H., Anderson, M. E., 36 ACS Paragon Plus Environment

Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Balasubramanian, C. L., Duniho, S. M., Leiske, C. I., Li, F., and Senter, P. D. (2014) Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat. Biotechnol. 32, 1059-62. (24)

(25)

Jackson, D., Atkinson, J., Guevara, C. I., Zhang, C., Kery, V., Moon, S. J., Virata, C., Yang, P., Lowe, C., Pinkstaff, J., et al. (2014) In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates. PLoS One 9, e83865. Behrens, C. R., Ha, E. H., Chinn, L. L., Bowers, S., Probst, G., Fitch-Bruhns, M., Monteon, J., Valdiosera, A., Bermudez, A., Liao-Chan, S., et al. (2015) Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional

(26)

(27) (28) (29)

(30)

(31)

(32)

Heterogeneous ADCs. Mol. Pharm. 12, 3986-98. Badescu, G., Bryant, P., Bird, M., Henseleit, K., Swierkosz, J., Parekh, V., Tommasi, R., Pawlisz, E., Jurlewicz, K., Farys, M., et al. (2014) Bridging disulfides for stable and defined antibody drug conjugates. Bioconjugate Chem. 25, 1124-36. Sugio, S., Kashima, A., Mochizuki, S., Noda, M., and Kobayashi, K. (1999) Crystal structure of human serum albumin at 2.5 A resolution. Protein Eng. 12, 439-46. Turell, L., Radi, R., and Alvarez, B. (2013) The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radic. Biol. Med. 65, 244-53. Alley, S. C., Benjamin, D. R., Jeffrey, S. C., Okeley, N. M., Meyer, D. L., Sanderson, R. J., and Senter, P. D. (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjugate Chem. 19, 759-65. Patterson, J. T., Asano, S., Li, X., Rader, C., and Barbas, C. F., 3rd. (2014) Improving the serum stability of site-specific antibody conjugates with sulfone linkers. Bioconjugate Chem. 25, 1402-7. Toda, N., Asano, S., and Barbas, C. F., 3rd. (2013) Rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: a serum-stable alternative to maleimide-based protein conjugation. Angew. Chem. Int. Ed. Engl. 52, 12592-6. Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L., and Buchwald, S. L. (2015) Organometallic palladium reagents for cysteine bioconjugation. Nature 526, 687-91.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

“For Table of Contents Only”


Page 38 of 38

One-Step Conjugation Method for Site-Specific AntibodyâDrug

Recommend Documents

One-Step Conjugation Method for Site-Specific AntibodyâDrug