DOI: 10.1002/cmdc.201900077
`
`Full Papers
`
`Characterization of Disulfide Bond Rebridged Fab–Drug
`Conjugates Prepared Using a Dual Maleimide
`Pyrrolobenzodiazepine Cytotoxic Payload
`Ben T. Ruddle+, Ryan Fleming+, Herren Wu, Changshou Gao,* and Nazzareno Dimasi*[a]
`
`We describe the characterization of antigen binding fragments
`(Fab)–drug conjugates prepared using a dual maleimide pyrro-
`lobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolo-
`benzodiazepine dimers, which are DNA cross-linkers, are a
`class of payloads used in antibody–drug conjugates (ADCs).
`SG3710 was designed to rebridge two adjacent cysteines, such
`as those that form the canonical interchain disulfide bond be-
`tween the light and heavy chain in Fab fragments. The re-
`bridging generated homogenous Fab conjugates, with a drug-
`to-Fab ratio of one, as demonstrated by the preparation of re-
`bridged Fabs derived from the anti-HER2 trastuzumab anti-
`
`body and from a negative control antibody both prepared
`using recombinant expression and papain digestion. The re-
`sulting anti-HER2 trastuzumab Fab-rebridged conjugate re-
`tained antigen binding, was stable in rat serum, and demon-
`strated potent and antigen-dependent cancer cell-killing abili-
`ty. Disulfide rebridging with SG3710 is a generic approach to
`prepare Fab–pyrrolobenzodiazepine dimer conjugates, which
`does not require the Fabs to be engineered for conjugation.
`Thus, SG3710 offers a flexible and straightforward platform for
`the controlled assembly of pyrrolobenzodiazepine dimer con-
`jugates from any Fab for oncology applications.
`
`Introduction
`
`Antibody drug conjugates (ADCs) are a class of drugs designed
`to deliver cytotoxic payloads to tumors, thus potentially in-
`creasing the therapeutic index of small-molecule cytotoxic
`drugs.[1] Four ADCs are approved for clinical use by the US
`Food and Drug Administration (FDA): brentuximab vedotin
`(Adcetris),[2] approved for Hodgkin lymphoma and systemic
`anaplastic large cell
`lymphoma;
`inotuzumab ozogamicin (Be-
`sponsa),[3] approved for relapsed or refractory B-cell precursor
`acute lymphoblastic leukemia; ado-trastuzumab emtansine
`(Kadcyla),[4] approved for human epidermal growth factor re-
`ceptor 2 (HER2)-positive metastatic breast cancer; and gentu-
`zumab ozogamicin (Mylotarg),[5] approved for CD33-positive
`acute myeloid leukemia. More than 65 ADCs are in various
`stages of clinical development, and numerous other ADCs are
`in late-stage preclinical characterization.[6]
`Most of the ADCs in preclinical and clinical development
`contain cytotoxic payloads that inhibit cell division, such as au-
`ristatin and maytansine.[7] Another class of ADCs contain DNA-
`damaging agents
`such as pyrrolobenzodiazepine dimers
`(PBD)[8] and duocarmycin analogues.[9] These ADCs are based
`on full-length antibodies. The molecular weight of these ADCs
`( 150 kDa) could pose multiple problems including poor
`
`[a] B. T. Ruddle,+ R. Fleming,+ Dr. H. Wu, Dr. C. Gao, Dr. N. Dimasi
`AstraZeneca, Antibody Discovery and Protein Engineering, One MedImmune
`Way, Gaithersburg, MD 20878 (USA)
`E-mail: gaoc@medimmune.com
`dimasin@medimmune.com
`[++] These authors contributed equally to this work.
`The ORCID identification number(s) for the author(s) of this article can
`be found under:
`https://doi.org/10.1002/cmdc.201900077.
`
`tumor penetration and uptake, increased systemic accumula-
`tion and slow clearance.[10, 11] In addition, the presence of the
`Fc region, while beneficial for effector functions and half-life in
`naked therapeutic antibodies, can cause Fc-mediated side ef-
`fects in ADCs.[12] Adcetris was shown to have no detectable
`complement-dependent cytotoxicity (CDC) and minimal anti-
`body-dependent cellular cytotoxicity (ADCC).[13] McDonagh
`et al. reported that comparable ADCs with or without Fc effec-
`tor functions could not conclusively demonstrate any benefit
`for Fc-enabled ADCs.[14] Gentuzumab ozogamicin (Mylotarg),[5]
`is based on IgG4, which has decreased Fc receptor binding.[15]
`More recently,
`IgG1 engineered for lack of Fc binding are
`being used as ADC scaffolds.[16, 17]
`Small fragments derived from IgGs or from other highly
`structured scaffolds derived from natural proteins have found
`use as ADCs.[10, 11, 18] Antibody fragments may be ideal formats
`as ADC cancer therapeutics because they could have higher
`tumor uptake, a better tumor-to-blood ratio, and faster clear-
`ance than full-length antibodies.[10, 11, 18] The former may be a
`desirable property in cases where toxicities to healthy tissues
`can increase with prolonged exposures. Furthermore,
`frag-
`ments do not have the Fc region, which could be beneficial to
`function related toxicities.[12]
`overcome potential effector
`Amongst the plethora of antibody formats, Fab fragments,
`which comprise the constant and variable domains of immu-
`noglobulins, have advantages over other antibody fragments,
`including: 1) high levels of recombinant expression, 2) straight-
`forward of preparation via papain digestion of full-length anti-
`bodies, 3) standard affinity chromatography purification from
`culture supernatant using anti-lambda, anti-kappa, or anti-
`CH1/CL media, 4) high thermostability, and 5) possession of
`
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`Figure 1. Structures of A) SG3249 and B) SG3710. Blue coding indicates the
`SG3199 warhead. Other key features of both payloads are shown. The N10
`position of the warhead is labeled in both SG3249 and SG3710. The molecu-
`lar weights are 1496 and 2408 Da for SG3249 and SG3710, respectively, and
`the respective logD values are 0.6 and 2.64.
`
`payload used in several ADCs in clinical development.[31–34]
`SG3710 contains two maleimide-(polyethylene glycol)8-valine-
`alanine-para-aminobenzoic acid linkers at each of the two sym-
`metrical N10 positions of the PBD (Figure 1 b). The warhead
`(i.e., the sequence-selective DNA minor-groove cross-linker) of
`SG3710 is SG3199 (depicted in blue in Figure 1),[35] which is
`also the warhead of tesirine (SG3249). SG3199 is released from
`SG3710, and from SG3249,
`in the lysosomal compartment
`upon cleavage by cathepsin B. After translocation to the nu-
`cleus and insertion in the DNA minor groove, an aminal bond
`is formed through nucleophilic attack of the N2 of a guanine
`base at the electrophilic C11 position on the SG3199.[35]
`The symmetrical structure of SG3710 offers two distinct and
`beneficial structural properties compared to SG3249. First, the
`additional (polyethylene glycol)8 linker decreases the hydro-
`phobicity of SG3710 versus SG3249. The SG3710 calculated
`logD value at pH 7.4 is 2.64, which is lower than the calculat-
`ed logD value of SG3249 (0.6). This can have a significant
`impact in improving the in vivo performance of ADCs[28] and
`FDCs prepared with SG3710, as it has been reported that ADCs
`with high hydrophobicity have poor pharmacokinetics and bio-
`distribution.[36, 37] Second, the release of the SG3199 warhead
`from SG3710 requires two cathepsin B cleavages,[38] whereas
`SG3249 requires only one cleavage.
`
`native thiols forming a solvent-accessible disulfide bond, which
`upon reduction can be used as attachment points for conjuga-
`tion.[19]
`Rebridging of the native interchain disulfide bond has been
`used as a strategy to prepare Fab–drug conjugates (FDCs) and
`ADCs.[20–27] For example, Godwin and colleagues[20, 21] developed
`a rebridging conjugation approach using a bis-sulfone linker–
`drug, in which interchain disulfide bonds in either full-length
`antibodies or Fab fragments were first partially reduced, fol-
`lowed by bis-alkylation to conjugate both thiols of the two
`native cysteines. The bis-sulfone linker carried a cytotoxic pay-
`load (monomethyl auristatin E), and the rebridging approach
`resulted in FDCs and ADCs that were more stable in human
`serum than conjugates prepared using non-rebridging malei-
`mide chemistry. Similarly, Behrens et al.[22] rebridged reduced
`interchain cysteines in antibodies using a dibromomaleimide
`monomethyl auristatin F payload, which resulted in the conju-
`gation of four cytotoxic drug–linkers per antibody. The result-
`ing ADCs were highly stable and had potent cytotoxicity
`against tumor cells. Caddick and colleagues contributed signifi-
`cantly over the past few years to the development of several
`rebridging approaches to prepare well-defined FDCs and
`ADCs.[23–26] Finally, rebridging of disulfide bonds through a
`thiol–yne reaction with a cyclooctyne has also been successful-
`ly applied to an antibody Fab.[27]
`Recently, we reported a novel dual-maleimide pyrrolobenzo-
`diazepine dimer cytotoxic payload (SG3710) that rebridged the
`cysteines at position 220 of the hinge domain of an engi-
`neered antibody, to produce a rebridged ADC with a drug-to-
`antibody ratio (DAR) of one.[28] The rebridged ADCs were
`highly resistant to payload loss in serum, had potent and selec-
`tive cytotoxicity, and importantly were tolerated in rats at
`twice the dose of equivalent non-rebridged ADCs but with
`DAR of two prepared using a mono-maleimide PBD (SG3249).
`Our data suggested that SG3710, which offers the opportunity
`to prepare IgG1-based ADCs with DAR of one, could be a strat-
`egy to improve the therapeutic index of PBD-based ADCs.[28]
`Here, we investigated whether SG3710 can be used to re-
`bridge the native cysteines that form the interchain disulfide
`bond between the light and heavy chain in Fabs. We demon-
`strated that SG3710 efficiently rebridged a Fab derived from
`the anti-HER2 trastuzumab antibody and a Fab from an isotype
`control antibody, and the resulting trastuzumab rebridged FDC
`was highly cytotoxic and specific against cancer cells in vitro.
`Furthermore, the rebridged Fabs were highly stable in rat
`serum. We also demonstrated that the SG3710 rebridging ap-
`proach is broadly applicable to Fabs produced either recombi-
`nantly or by papain digestion of full-length antibodies. The re-
`bridged FDC platform described here may offer an alternative
`to full-length ADCs for oncology therapeutics that use PBDs.
`
`Results and Discussion
`
`Structural characteristics of SG3710
`
`The dual-maleimide PBD SG3710[28] was synthesized using
`starting material from tesirine (SG3249, Figure 1 a),[29, 30] a PBD
`
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`Preparation of Fabs from trastuzumab and from an isotype
`control antibody
`
`Fabs from the anti-HER2 antibody trastuzumab and from the
`isotype control antibody NIP228 were prepared by papain di-
`gestion using full-length antibodies and by transient expres-
`sion. Trastuzumab and NIP228 antibodies were transiently ex-
`pressed in Chinese hamster ovary (CHO) cells with yields of
`300 mg L1 after 14 days of transient expression. Expression
`levels were quantified by using a protein A quantification
`method.[39, 40] Purification was carried out by protein A affinity
`chromatography, which resulted in 99 % monomeric content
`for both antibodies (Figure 2 a). Fabs were prepared by papain
`
`Full Papers
`
`digestion of antibodies. Digestion was monitored by size-exclu-
`sion chromatography (SEC, data not shown). After approxi-
`mately 80 % digestion, both trastuzumab and NIP228 Fabs
`were purified by anti-kappa affinity chromatography. SEC of
`the purified Fabs showed monomeric content for both Fabs of
`approximately 80 %, with the presence of high-molecular-
`weight aggregates, undigested antibodies and fragments (Fig-
`ure 2 b). For the transient expression, the Fabs were cloned
`into a MedImmune mammalian expression vector,[39, 40] and ex-
`pression was carried out in CHO cells for 14 days, resulting in
`250 and 150 mg L1 for trastuzumab Fab and NIP228 Fab, re-
`spectively. The Fabs were purified from the culture medium by
`anti-kappa affinity chromatography. Both recombinant Fabs
`had a monomeric content of 95 %, with minor amounts of ag-
`gregates and fragments (Figure 2 c). The Fabs prepared by
`papain digestion and by transient expression were used direct-
`ly for rebridging without further purification.
`
`Conjugation process for preparing rebridged FDCs
`
`A schematic representation of the rebridging process used in
`this study is shown in Figure 3. Conjugation of SG3710 to the
`cysteines forming the interchain disulfide bond between the
`light and heavy chains in Fabs occurs via thiol-Michael addi-
`tion.[41] The Fabs prepared using papain digestion contain four
`additional
`residues, DKTH, at
`the C terminus of
`the CH1
`domain. Those residues come after the cysteine at position
`
`Figure 2. SEC traces for A) antibodies, B) Fabs prepared using papain diges-
`tion from antibodies, and C) Fabs prepared using transient expression. The
`percentage of monomeric content is shown. The arrows show fragments,
`aggregates and/or full-length antibodies remaining after papain digestion.
`
`Figure 3. Experimental procedure used to rebridge the cysteines that form
`the interchain disulfide bonds in Fabs. The Fabs prepared using papain di-
`gestion contain four additional residues (DKTH) after the CH1 C-terminal cys-
`teine at position 220. The interchain disulfide bond in Fabs is formed by the
`CH1 cysteine at position 220 and the light-chain cysteine at position 214.
`
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`220 that forms the interchain disulfide bond with the C-termi-
`nal cysteine at position 214 of the light chain. The Fabs pre-
`pared using recombinant expression do not have the addition-
`al four residues after the CH1 C-terminal cysteine at position
`220.
`
`Rebridging of trastuzumab and NIP228 Fabs prepared using
`papain digestion
`
`The trastuzumab and NIP228 Fabs prepared using papain di-
`gestion were rebridged by reducing the interchain disulfide
`bridge between the light and heavy chains, followed by conju-
`gation with SG3710 (Figure 3). The reducing conjugation reac-
`tions were optimized to be carried out sequentially without
`the need to remove excess reducing reagent. Reduced re-
`versed-phase
`liquid chromatography mass
`spectrometry
`(rRPLC–MS) showed that rebridging produced nearly homoge-
`
`nous conjugates for both trastuzumab and NIP228 Fabs, as
`demonstrated by a very minor amount of non-rebridged light
`and heavy chains (Figure 4). The deconvoluted mass spectrom-
`etry data show the presence of unidentified molecular weight
`species and the minor presence of non-rebridged light and
`heavy chains (Figure 4, arrows). These Fabs used for the re-
`bridging after papain digestion were about 85 % monomer,
`which could explain the presence of non-rebridged light and
`heavy chains and the unidentified mass species in the rRPLC–
`MS data (Figure 4 arrows).
`
`Rebridging of trastuzumab and NIP228 Fabs prepared by
`transient expression
`
`The Fabs prepared by recombinant expression were rebridged
`by using a process similar to that of the papain-digested Fabs.
`The rebridging reaction analyzed by rRPLC–MS revealed high
`
`Figure 4. Rebridging of Fabs prepared using papain digestion analyzed by rRPLC–MS. Shown are the deconvoluted mass spectra of A) trastuzumab Fab, B) re-
`bridged trastuzumab Fab, C) NIP228 Fab, and D) rebridged NIP228 Fab. The molecular weights of the light and heavy chains, and of the rebridged Fabs are
`shown. The cartoons represent the reduced light and heavy chain (A, C) and the rebridged Fabs (B, D). The calculated molecular weight of the papain-digested
`trastuzumab Fab is 47667 Da (23439 Da molecular weight light chain + 24228 Da molecular weight heavy chain). The determined molecular weight of the re-
`bridged trastuzumab Fab is 50075, which is 2408 Da more than the non-rebridged Fab, which correspond to the molecular weight of SG3710 (Figure 1). The
`molecular weight of the papain-digested NIP228 Fab is 47060 Da (23188 Da molecular weight light chain + 23872 Da molecular weight heavy chain). The de-
`termined molecular weight of the rebridged NIP228 Fab is 49468, which is 2408 Da more than the non-rebridged Fab, which correspond to the molecular
`weight of SG3710 (Figure 1). The arrows in A and C indicate unidentified molecular weight species. The arrows in B and D indicate reduced light and heavy
`chains and unidentified mass species.
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`Figure 5. Rebridging of Fabs prepared using transient expression analyzed by rRPLC–MS: A) Trastuzumab Fab, B) rebridged trastuzumab Fab, C) NIP228 Fab,
`D) rebridged NIP228 Fab. The molecular weight of the respective light and heavy chains, and of the rebridged Fabs are shown. The cartoons represent the re-
`duced light and heavy chain and the rebridged Fabs. The calculated molecular weight of the recombinantly produced trastuzumab Fab is 47186 Da
`(23439 Da molecular weight light chain + 23747 Da molecular weight heavy chain). The determined molecular weight of the rebridged trastuzumab Fab is
`49594, which is 2408 Da more than the non-rebridged Fab, which correspond to the molecular weight of SG3710 (Figure 1). The calculated molecular weight
`of the recombinantly produced NIP228 Fab is 46578 Da (23188 Da molecular weight light chain + 23390 Da molecular weight heavy chain). The determined
`molecular weight of the rebridged NIP228 Fab is 48986, which is 2408 Da more than the non-rebridged Fab, which correspond to the molecular weight of
`SG3710 (Figure 1). The molecular weight difference between the Fabs prepared using papain digestion shown in Figure 4, and those prepared using transient
`expression shown here is due to the residues DKTH that remain on the heavy chains of the Fabs after papain digestion. As in Figure 4, the arrows in A and C
`indicate unidentified molecular weight species. The arrows in B and D indicate reduced light and heavy chains and unidentified mass species.
`
`efficiency of rebridging, because there is very minimal measur-
`able amounts of reduced light and heavy chain in the rebridg-
`ing reaction (Figure 5). As for the rebridged Fabs prepared
`from papain digestion, the rRPLC–MS data showed the pres-
`ence of unidentified molecular mass species and very minimal
`amounts of reduced light and heavy chains (Figure 5 arrow).
`
`Analytical characterization of the rebridged FDCs
`
`After the rebridging was demonstrated by rRPLC–MS (Figures 4
`and 5), and the FDCs purified by preparative hydrophobic in-
`teraction chromatography (HIC, data not shown) to remove
`free SG3710 and other contaminants, the FDCs were further
`characterized by several analytical orthogonal methods.[16, 17, 28]
`SEC demonstrated that the purified rebridged FDCs were more
`than 97 % monomeric (Figure 6 A, B, G, H). rRPLC confirmed
`near homogeneity of the rebridged FDCs because there was
`
`very minimal presence of reduced light and heavy chains (Fig-
`ure 6 C, D, I, J). HIC further confirmed the high degree of ho-
`mogeneity of the rebridged FDCs because it showed no de-
`tectable non-conjugated Fabs (Figure 6 E, F, K, L). The analytical
`characterization demonstrated that rebridging of the cysteine
`forming the interchain disulfide bridge in Fabs results in homo-
`genous FDCs for Fabs prepared using papain digestion and for
`Fabs prepared using recombinant expression.
`
`In vitro rat serum stability of the rebridged FDCs
`
`The stability of the rebridged FDCs was assessed by incubating
`the FDCs in rat serum for 15 days at 37 8C. The FDCs were af-
`finity purified from the rat serum at the start of the incubation,
`which was used as time zero reference, and after 15 days incu-
`bation, and analyzed by rRPLC–MS (Figure 7). The stability in
`serum was determined by analyzing the mass peak signals of
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`Figure 6. Analytical characterization of the FDCs. Panels A–F show the analytical characterization for the FDCs prepared using papain digestion. Panels G–L
`show the analytical characterization for the FDCs prepared using transient expression. Panels A, B, G, and H show SEC data. Panel C, D, I, and J show rRPLC
`data. Panel E, F, K, and L show HIC data. An absorbance of 280 nm was used for SEC and HIC, and an absorbance of 214 nm was used for rRPLC. The FDCs
`were more than 97 % monomeric as demonstrated by SEC. HC and LC denote the Fab heavy and light chains, respectively.
`
`the rebridged species at day 15 compared with the signals at
`day zero. The FDCs were purified from the rat serum using an
`
`anti-human IgG F(ab’)2 antibody. This antibody has low cross-
`reactivity to rat serum proteins, except that an unknown pro-
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`Figure 7. Stability of the FDCs in rat serum prepared using transient expression. FDCs were incubated in rat serum for 15 days and then affinity purified using
`a goat anti-human IgG F(ab’)2 antibody. The affinity-purified FDCs were analyzed by rRPLC–MS, and their stability was analyzed by comparing the mass peak
`signals for the rebridged species at day 15 versus the rebridged mass peaks at day zero. The rebridged FDCs mass peak remined unchanged throughout the
`studies. An unidentified rat protein was co-purified (peak identified with an asterisk).
`
`tein was immunopurified (Figure 7). This analysis revealed that
`the rebridged FDCs were highly stable in rat serum throughout
`the duration of the studies (Figure 7).
`
`In vitro cytotoxicity of the rebridged FDCs
`
`The in vitro cytotoxicity of the FDCs was tested against HER2
`breast cancer cell
`lines SKBR-3 (HER2 3 +) and MDA-MB-453
`
`(HER2 2 +), which have high and medium levels of HER2 ex-
`pression, respectively. MDA-MB-468, a breast cancer cell
`line
`that does not express HER2, was used as negative control. As
`ADC positive control, trastuzumab site-specific conjugated to
`two SG3249 was used. The trastuzumab rebridged FDC
`showed potent in vitro cytotoxicity (Figure 8 A, B, C, E, F),
`whereas no cytotoxicity was observed when the FDCs were
`tested against the HER2-negative cell line (Figure 8 D). The neg-
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`Figure 8. In vitro cytotoxicity of FDCs and ADC (trastuzumab site-specifically conjugated to two SG3249) against breast cancer cell lines SKBR-3 and MDA-MB-
`453, which express high and low levels of HER2, respectively. The MDA-MB-468 breast cancer cell line that does not express HER2 was used as negative con-
`trol. IC50 values for trastuzumab FDC and ADC are shown. Each graph represents individual experiments, each performed in triplicate. Panel B is a biological
`replicate of panel A. The x-axis represents the logarithm of the protein concentration.
`
`ative control NIP228 rebridged FDC had no cytotoxicity against
`SKBR-3 cancer cells (Figure 8 A). The trastuzumab FDC had sim-
`ilar potency to trastuzumab ADC in the high-HER2-expressing
`SKBR-3 cell line (Figure 8 E). A 30-fold difference in potency be-
`tween trastuzumab FDC versus trastuzumab ADC was ob-
`served in the low-HER2-expressing breast cancer cell line MDA-
`MB-453 (Figure 8 F).
`
`Conclusions
`
`The development of ADCs has evolved greatly over the past
`few years, resulting in four ADCs approved for the treatment
`of solid and hematologic tumors.[3, 4, 5] However, technological
`challenges still need to be overcome to improve the therapeu-
`tic index (TI, defined as the maximum tolerated dose divided
`by the minimum efficacious dose) of ADCs, particularly those
`used to treat solid tumors and those prepared using PBDs.[17]
`Several efforts have recently been made to improve the TI of
`
`including 1) site-specific conjugation, which offers pre-
`ADCs,
`cise drug loading and optimal serum stability,[1] 2) fractionated
`doses of PBD ADCs, which maintain antitumor activity and
`have greater tolerability than single high doses,[18] and 3) im-
`provements in tumor penetration of small-fragment ADCs.[19]
`Recently we reported that lowering the number of drugs
`per antibody and using a rebridging conjugation strategy with
`a dual maleimide PBD (SG3710) could be a strategy to improve
`the TI of an anti-HER2 antibody.[28] Herein, we further expanded
`the utility of the SG3710 payload to rebridge the native cys-
`teines that form the interchain disulfide bonds between the
`light and heavy chains in Fabs. The rebridging approach we
`developed can be used for Fabs prepared from full-length anti-
`bodies using papain digestion or for Fabs prepared using tran-
`sient expression. Site-specific rebridging of
`the Fabs with
`SG3710 was successfully achieved as demonstrated by several
`orthogonal analytical methods. Our previous studies showed
`that full-length antibodies rebridged at the hinge with SG3710
`
`ChemMedChem 2019, 14, 1185 – 1195
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`www.chemmedchem.org
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`1192
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` 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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`were highly stable in mouse serum.[28] The Fabs rebridged with
`SG3710 maintained stability in rat serum, as shown by the lack
`of appreciable drug loss after 15 days of incubation. Having
`demonstrated that the rebridged FDCs were stable in serum,
`we determined that the rebridged anti-HER2 FDC had potent
`and selective in vitro cytotoxicity against breast cancer cells ex-
`pressing various levels of HER2. These data demonstrated that
`the anti-HER2 FDC maintained binding specificity to HER2 and
`was efficiently internalized into target-positive cells.
`In the
`HER2-overexpressing cell
`line SKRB-3,
`the anti-HER2 FDC
`showed similar potency of the trastuzumab ADC site-specifical-
`ly conjugated with two PBDs. This is not surprising because
`high-expression of the HER2 receptors in this cell
`line pro-
`motes high internalization regardless of the difference in avidi-
`ty between the FDC and ADC, and because of the high poten-
`cy of the warhead SG3199 common to both SG3249 and
`SG3710. In the breast cancer cell line MDA-MB0-453, which ex-
`presses low levels of HER2, a marked difference in potency was
`observed between the trastuzumab FDC and ADC. In fact, tras-
`tuzumab ADC had similar potency in high- and low-HER2-ex-
`pressing cancer cell lines (Figure 8 E, F), whereas a 30-fold dif-
`ference was observed for the trastuzumab FDC between the
`high- and low-HER2-expressing cancer cell lines (Figure 8 C, E,
`F).
`
`In conclusion, this is the first report of the preparation of
`FDCs using a rebridging strategy with a payload, SG3710, that
`has symmetrical linkers for conjugation and a warhead for cy-
`totoxicity. The robust synthesis of SG3710, a simple rebridging
`process, and the negligible aggregate formation during the re-
`bridging indicate that this novel class of rebridged FDCs could
`be manufactured. Moreover, the potential beneficial properties
`offered by small-fragment ADCs, such as superior tumor pene-
`tration and more rapid systemic clearance, warrant further in-
`vestigation of the FDC platform described herein.
`
`Experimental Section
`
`General: All
`reagents were purchased from Sigma–Aldrich or
`Thermo Fischer Scientific unless otherwise stated. The payload
`used for this study, SG3710, was 95 % pure, and its synthesis was
`previously reported.[28] Solvents used for the analytical characteriza-
`tion of antibodies, Fabs, FDS, and ADCs were purchased from JT
`Baker or prepared at MedImmune. Size-exclusion chromatography
`(SEC), reduced reversed-phase liquid chromatography (rRPLC) and
`hydrophobic interaction chromatography (HIC) were carried out
`with an Agilent 1290 Infinity II HPLC system equipped with an au-
`tosampler and diode array detector. ChemStation software (Agi-
`lent) was used to control the analytics systems and analyze the
`data. An absorbance of 280 nm was used to detect antibodies,
`Fabs, and FDCs for SEC and HIC, while an absorbance of 214 nm
`was used for rRPLC. Antibodies, Fabs, and FDCs were quantified
`using a Nanodrop (Thermo Fischer Scientific), an absorbance of
`280 nm and an extinction coefficient of 1.6 for antibodies and
`Fabs, and 1.4 for FDCs. LogD values of SG3710 and SG3249 were
`calculated at pH 7.4 using ACD ChemSketch (ACD/LABS).
`
`Antibodies and Fabs: An anti-HER2 antibody, derived from trastu-
`zumab, and a negative control antibody, NIP228 (MedImmune),
`were used as the full-length antibodies to prepare the Fabs. Molec-
`
`Full Papers
`
`ular biology experiments were carried out as described previous-
`ly.[15]
`
`Transient expression of antibodies and Fabs: Antibodies and
`Fabs were cloned into a proprietary MedImmune mammalian ex-
`pression vector,[40] and transient expression was carried out using
`suspension-adapted CHO cells. Expression was carried out for
`14 days using a MedImmune proprietary culture medium. The ex-
`pression level in the culture supernatant was determined using a
`protein A binding method described by Dimasi et al.[40] After ex-
`pression, the cells were discarded by centrifugation and the culture
`medium was filtered. The antibodies were affinity purified with
`protein A (GE Healthcare), and the Fabs with anti-kappa (GE
`Healthcare) using the manufacturer’s specifications. The purified
`antibodies and Fabs were formulated in 1 ” PBS, 1 mm EDTA at
`pH 7.2 and stored at 4 8C until use.
`
`Conjugation: The Fabs prepared using papain digestion were re-
`duced using 10 equivalents of dithiothreitol (DTT, Sigma–Aldrich)
`at 37 8C with shaking for 30 min, followed by buffer exchange in
`1 ” PBS, pH 7.2 to remove excess DTT. Before conjugation these
`Fabs were reduced again with two-fold equivalents of tris(2-car-
`boxyethyl)phosphine (TCEP) at 37 8C with shaking for 1 h. The Fabs
`prepared using transient expression were reduced only once using
`two-fold equivalents of TCEP at 37 8C with shaking for 1 h. The re-
`duced Fabs were then used for conjugation using two-fold equiva-
`lents of SG3710 prepared in 100 % dimethyl sulfoxide (10 % final
`DMSO concentration, Sigma–Aldrich). The conjugation was carried
`out with shaking at room temperature for 1 h. Trastuzumab site-
`specifically conjugated to two PBDs (SG3249) was prepared and
`characterized as described previously.[17, 34]
`