Engineering Antibody Reactivity for Efficient Derivatization to Generate

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Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates Kaustav Biswas, Thomas E Nixey, Justin K. Murray, James R Falsey, Li Yin, Hantao Liu, Jacinthe Gingras, Brian E. Hall, Brad Herberich, Jerry Ryan Holder, Hongyan Li, Joseph Ligutti, Min-Hwa Jasmine Lin, Dong Liu, Brian D Soriano, Marcus Soto, Linh Tran, Christopher M. Tegley, Anrou Zou, Kannan Gunasekaran, Bryan D. Moyer, Liz Doherty, and Les P Miranda ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00542 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Chemical Biology

Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates Kaustav Biswas,*,† Thomas E. Nixey,† Justin K. Murray,† James R. Falsey,† Li Yin,† Hantao Liu,‡ Jacinthe Gingras,∆ Brian E. Hall,# Brad Herberich,† Jerry Ryan Holder,† Hongyan Li,§ Joseph Ligutti,‡ Min-Hwa Jasmine Lin,# Dong Liu,‡ Brian D. Soriano,† Marcus Soto,§ Linh Tran,§ Christopher M. Tegley,† Anrou Zou,‡ Kannan Gunasekaran,† Bryan D. Moyer,*,‡ Liz Doherty,† and Les P. Miranda† †

Therapeutic Discovery, ‡Neuroscience, and §Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA and #Therapeutic Discovery, ∆ Neuroscience, #Pharmacokinetics and Drug Metabolism, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA macromolecule design, bioconjugate, reactivity optimization, NaV1.7, voltage-gated sodium channel, half-life extension, nerve, biodistribution *

To whom correspondence should be addressed:

Dr. Kaustav Biswas Amgen One Amgen Center Drive MS 29-M-B Thousand Oaks, CA 91302 Phone: 805-447-4836 [email protected]

Dr. Bryan D. Moyer Amgen One Amgen Center Drive MS 29-2-B Thousand Oaks, CA 91302 Phone: 805- 313-5495 [email protected]

ACS Paragon Plus Environment

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ABSTRACT The voltage-gated sodium channel NaV1.7 is a genetically-validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain, however functional NaV1.7 targeting antibodies are not known. In this report we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of thirteen potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a non-conjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study outlines the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.


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Chronic pain is a critical unmet medical need afflicting millions of individuals with an economic impact that rivals heart disease, cancer, and diabetes.1 Neuropathic pain due to a lesion or disease of the somatosensory system is particularly grievous and estimated to impact between seven and ten percent of the population.2 Current treatment options are limited by safety and tolerability, only benefiting one in four individuals.3,4 The over-prescription of opioids for chronic pain highlights the necessity for new therapeutics targeting non-opioid pathways for pain relief.

The voltage-gated sodium channel NaV1.7 has been the subject of extensive research as a target for developing new therapies for pain, with validation from human gain-of-function and loss-of-function genetic mutations in SCN9A, the gene encoding the NaV1.7 protein.5 NaV1.7 channelopathies include congenital insensitivity to pain, where channel function is impaired, and erythromelalgia, paroxysmal extreme pain disorder and small fiber neuropathy, where channels are hyperactive leading to ectopic nociceptor action potential firing.6 A challenge in developing NaV1.7 inhibitors is engineering NaV subtype selectivity against related sodium channels including NaV1.4 and NaV1.5 that play critical roles in skeletal muscle and heart function, respectively.7,8 A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain, however the only reported NaV1.7 targeting antibody9 could not be reproduced.10 We pursued an alternate approach to generate a large molecule blocker of NaV1.7 function. Known ion-channel blockers include naturally occurring peptide inhibitors of NaV1.7 identified from the venom of poisonous species like spiders and cone snails.8 We discovered the peptide GpTx-1 from venom of the Chilean tarantula Grammostola porteri using a venom screen and hit-


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identification process.11 Detailed structure activity relationship (SAR) studies led to GpTx-1 analogs with high potency and selectivity against other NaV channels.12 We now considered a hybrid large molecule construct employing a bifunctional design comprised of a GpTx-1 targeting warhead tethered by chemical conjugation to a carrier protein to address the lack of targeting antibodies that inhibit NaV1.7. This approach would modify the in vivo properties of the peptide as well. Disulfide-rich inhibitory cystine knot motif peptides like GpTx-1 possess high proteolytic stability in plasma.11,13 However, due to low molecular weight (3.5 – 4 kDa), they are subject to rapid glomerular filtration and exhibit plasma half-lives of less than 2h.14 Additionally, distribution of unmodified peptides from the vasculature into compartments expressing a pain target such as NaV1.7 is not normally preserved when applying peptide halflife extension methodologies like PEG-ylation.15 Antibody conjugation could therefore also potentially improve the circulating half-lives and alter biodistribution of these potent NaV1.7 blockers via the neonatal Fc receptor (FcRn) recycling mechanism.16 Multiple approaches to peptide derivatization are known, including lipidation and conjugation to carrier proteins such as albumin and Fc domains of antibodies.17 Peptides expressed as fusions with antibodies or Fc domains have demonstrated pharmacodynamic effects due to half-life extension (HLE).18 To prepare GpTx-1 peptide antibody conjugates, we selected a site-specific conjugation approach initially developed by Junutula et al., with reaction of a peptide-linker moiety to a cysteine engineered antibody, producing homogenous conjugates loaded with two copies of peptide.19,20 This study extends the application of the cysteine derivatization technology to a non-oncology target, the inhibition of the NaV1.7 ion channel. Herein, we report the design and chemistry optimization of site-specific antibody conjugates of GpTx-1 analog peptides, confirmation of structure, functional inhibition of NaV1.7 currents in


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HEK293 and neuronal cells by electrophysiology, and mouse pharmacokinetic studies demonstrating HLE. Furthermore, analysis of conjugate biodistribution using fluorescencebased imaging demonstrated differential distribution to sites of NaV1.7 expression in nerves of wild-type (WT) but not NaV1.7 knock-out (KO) mice. Through these studies, we illustrate an alternate strategy for selectively inhibiting NaV1.7 in cells with a large molecule combining FcRn-based antibody recycling and ion-channel targeting by the GpTx-1 peptide.

RESULTS AND DISCUSSION Conjugate Design and Synthesis The site of attachment of a warhead to an antibody scaffold can significantly impact the stability, activity, and pharmacokinetic properties of conjugates.21,22 We adopted the strategy of engineering cysteines into the antibody for chemical conjugation to a NaV1.7 inhibitory peptide. We prepared a panel of carrier antibodies using a non-targeting anti-2,4-dinitrophenol (αDNP) human IgG1 antibody scaffold, each with a different surface-exposed residue mutated to a cysteine. The criteria for selection included residues with calculated accessible surface areas greater than 20 Å, and excluded the antibody complimentarity-determining region and effector binding domains as well as glycine and proline residues replacing which can potentially alter protein structures (Figure 1a).

For a synthetic protocol, we desired flexibility with respect to three molecular inputs, i.e. peptide, linker, and antibody, to enable independent optimization of each. In addition, the chemistry had to be orthogonal to all reactive functional groups contained within peptide and


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protein moieties. As a first step, we identified positions on the peptide that tolerate incorporation of linkers with minimal loss in NaV1.7 inhibitory activity.23 The potent and selective GpTx-1 analog [Ala5,Phe6,Pra13,Leu26,Arg28]GpTx-1(1–34), peptide 1, with a derivatization site at position 13 was selected for study. We previously reported reaction of 1 with azide-containing PEG11 linker via a copper-catalyzed 1,3-dipolar cycloaddition reaction to generate a triazole linkage without affecting integrity of peptide disulfides.23 Here we rendered the linker bifunctional by adding a thiol-reactive moiety for subsequent conjugation to the engineered antibody cysteine. We selected bromoacetamide as the second reactive handle due to its lower propensity to hydrolysis (relative to iodoacetamide) and increased stability of the resulting thioether (relative to a maleimide). The two-step process of coupling linker to peptide and conjugating peptide-linker to antibody allowed variation of each constituent (Figure 1b).

Figure 1. (A) Representation of antibody scaffold with thirteen cysteine mutation sites evaluated in this study highlighted in red. (B) Conjugate synthesis protocol with modular control over peptide, linker, and antibody site: (a) PEG11 linker attachment to peptide via Huisgen cycloaddition (the alkyne, azide and resulting triazole are highlighted); (b) free, reactive thiol 4 generated from engineered, capped cysteine (E384C is exemplified) of antibody 3 via partial


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reduction or redox conditions (R, R’ = cysteine or glutathione) and (c) conjugation of peptidelinker bromoacetamide 2 at engineered cysteine.

Engineered cysteines on expressed antibodies are capped with endogenous thiols, such as glutathione or cysteine, which must be removed prior to alkylation.19 We investigated two conjugation methods: partial reduction and reduction/oxidation (redox) and reactivity of the designed cysteine mutants by using a model iodo-biotin alkylating agent, N-iodoacetyl-Nbiotinylhexinylenediamine (Figure 2a,b and S2). Under partial reduction conditions, varying equivalents of a reductant, e.g. the water soluble phosphine TCEP (tris(2carboxyethyl)phosphine)), were added, leading to reduction of only the two engineered cysteines on the antibody surface, leaving the internal inter- and intra-chain disulfide bonds intact. After removal of excess TCEP via desalting, addition of alkylation reagent afforded the conjugate and conversion was estimated by liquid chromatography time-of-flight mass spectroscopy (LC-TOFMS). Some sites were alkylated with higher efficiency than others (Figure 2a). Though straightforward, it was difficult to drive partial reduction reactions to greater than 80% conversion using excess reductant or reaction time without observing over-alkylation of native cysteines, particularly in the more solvent exposed residues forming the hinge disulfides (data not shown). In the redox paradigm, first an excess of TCEP was used to reduce both the engineered and inter-chain disulfides. This was followed by re-oxidation with a mild oxidant, dehydroascorbic acid (DHAA), to reform the inter-chain disulfides, after which only the engineered cysteine was available for alkylation with iodo-biotin. Higher conversions were observed in the redox protocol, with clear differences being observed in reactivity across the various sites (Figure 2b). We selected the redox protocol for synthesis due to reliably higher yields of desired products and lower propensity for by-product formation. Ten cysteine mutants


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were selected from the model alkylation reaction with iodo-biotin for a redox screen using the GpTx-1 peptide-linker bromoacetamide 2 (Figure 2c). For each individual conjugation site, the optimal TCEP:DHAA ratio identified in Figure 2b was used for peptide conjugation using the redox protocol. Reactivity was similar or lower than with iodo-biotin, with a few sites failing to couple efficiently with the more bulkier peptide reagent. The E384C mutation in the Fc CH2 domain (Figure S1) provided an engineered antibody that demonstrated optimum peptide alkylation efficiency using the redox protocol.

Figure 2. Alkylation optimization screen with cysteine engineered antibodies monitored by LCTOF-MS of the reduced and deglycosylated conjugate, shown as a ratio of desired monoalkylated peak to sum of unreacted, mono and bis-alkylated peaks in the MS spectrum. (A) Partial reduction screen with iodo-biotin reagent using varying amounts of TCEP per engineered


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cysteine. (B) Redox screen with iodo-biotin reagent using varying ratios of TCEP and DHAA per engineered cysteine. (C) Comparison of alkylation with iodo-biotin and GpTx-1 peptidelinker 2 employing the redox protocol using optimal TCEP/DHAA conditions identified for each site in 2b.

The E384C antibody was reacted with peptide-linker 2 to afford conjugate 5 on larger scale (Supporting Information). Conjugation using the redox protocol gave a mixture of the desired Drug Antibody Ratio (DAR) 2, monovalent DAR1 and unreacted DAR0 species in 74%, 14% and 16% conversion, respectively (Figure S4). After purification, we obtained pure divalent conjugate in 72% isolated yield (Figure S5-S7). The fidelity of conjugation of peptide-linker 2 to engineered cysteines on the E384C antibody was investigated by mass spectroscopic analysis of intact and reduced conjugate as well as by peptide mapping of conjugated and non-conjugated E384C antibody (Figure S8a-c ). Mass data for all species indicated that there was a single conjugated cysteine on the Fc CH2 domain where the engineered E384C resides, and the conjugate displayed a DAR of 2. Analysis of the peptide map data confirmed conjugation of the peptide to the E384C residue of the heavy chain.

Effect of conjugate architecture on functional activity Conjugate 5, composed of peptide 1, a PEG11 linker and the αDNP E384C antibody, was tested in a whole cell patch clamp electrophysiology assay on an automated PatchXpress® (PX) system using NaV1.7 stably transfected HEK293 cells.11 Conjugate 5 had an IC50 value of 250 nM (Table 1). The peptide-linker moiety was next conjugated to an engineered cysteine on a carrier protein comprised of only the Fc portion of the antibody. The Fc conjugate 6 blocked


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NaV1.7 with an IC50 value of 190 nM, similar to conjugate 5. We also tested the monovalent antibody conjugate 7 with only one heavy chain cysteine linked to the NaV1.7 inhibitory peptide, isolated as a byproduct of conjugation (DAR = 1, Figure 3c). This molecule was a weak blocker of NaV1.7, with an IC50 of 1,900 nM, demonstrating benefit of divalent architecture. We next examined four other conjugate design parameters: linker length, peptide warhead presentation and loading and site of attachment on the antibody. Increasing or decreasing the PEG linker chain length to three or 23 units in the conjugate had a limited overall effect on NaV1.7 inhibition (compounds 8 – 9). The effects of peptide copy number was investigated using a branched PEG23 linker designed to load two copies of peptide on each engineered cysteine (Figure 3d and S9). Attaching one copy of dimeric peptide-linker led to “asymmetric” conjugate 10 (Figure 3e) with a similar NaV1.7 IC50 as the corresponding “symmetric” PEG23 conjugate 9. Incorporating two units of dimeric linker afforded conjugate 11 with four copies of the peptide loaded onto the antibody (Figure 3f) and resulted in a five-fold increase in potency (IC50 = 57 nM). Analysis of ten conjugates where the peptide is connected to different cysteine mutants (5, 12–20) revealed a site dependence of potency, with 10-fold variation between the most potent (D88C conjugate 15, IC50 = 59 nM) and least potent (D377C conjugate 20, IC50 = 599 nM). Thus, varying peptide load and site of conjugation can modulate potency of NaV1.7 inhibition.


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Figure 3. Models of conjugate architectures. (A) Symmetric divalent conjugate, e.g. 5. The site of conjugation on the antibody, E384C, is highlighted. (B) Divalent Fc conjugate 6. (C) Monovalent conjugate 7. (D) Dimeric PEG23 linker for conjugates 10 and 11. (E) Asymmetric conjugate with 2 copies of peptide, 10. (F) Conjugate 11 with 4 copies of peptide.

Table 1. NaV1.7 IC50 values for conjugates 5 – 20 in PatchXpress electrophysiology assay Compound

Cys mutant

Linker

Peptide

NaV1.7 PXa

Loading

IC50 (nM)

5

E384C

PEG11

2

250

6

E384Cb

PEG11

2

190

7

E384C

PEG11

1

1,897

8

E384C

PEG3

2

321

9

E384C

PEG23

2

208

10

E384C

Bis-PEG23

2

305

11

E384C

Bis-PEG23

4

57

12

N526C

PEG11

2

325


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13

T487C

PEG11

2

78

14

E99C

PEG11

2

76

15

D88C

PEG11

2

59

16

S156C

PEG11

2

176

17

A154C

PEG11

2

185

18

E473C

PEG11

2

263

19

E189C

PEG11

2

184

20

D377C

PEG11

2

599

a

At least four different concentrations of test compound spanning four full log units were applied individually. % inhibition as a function of compound concentration was pooled from at least n = 14 compound additions to fit each IC50, similar to previously reported electrophysiology data.11,12 b Fc version of E384C Ab.

Conjugate 5 electrophysiology To characterize these new constructs for functional inhibition of NaV channels by electrophysiology, conjugate 5 was selected for initial profiling studies. We evaluated selectivity against representatives of two classes of sodium channels based on inhibition by the marine toxin tetrodotoxin (TTX). Tested channels included some sensitive to inhibition by TTX like NaV1.7 and NaV1.4 present in skeletal muscle and a channel resistant to TTX, NaV1.5, present in cardiac myocytes, and compared the potency of the conjugate with the parent peptide. Conjugate 5 was 30-fold less potent relative to naked peptide 1 with an IC50 value of 8.5 nM23 and 6-fold less potent relative to peptide-PEG11 control 21 (Figure S2), which had an IC50 value of 39 nM. Hence potency is impacted both by linker as well as antibody. Similar to the selectivity profile attained from peptide optimization,11,23 conjugate 5 did not potently block NaV currents from these other isoforms when expressed in heterologous HEK293 cells and evaluated on a PX platform, demonstrating selectivity ≥ 30-fold over NaV1.7 block (Figure 4c). We examined


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conjugate 5 using manual whole cell patch clamp electrophysiology,11 and determined the human NaV1.7 IC50 to be 297 nM, which is similar to the value obtained by automated electrophysiology (Figure 4a,b). Similarly, in native mouse dorsal root ganglion (DRG) neurons, conjugate 5 was a potent inhibitor of TTX-sensitive currents mediated in part by native NaV1.724 with an IC50 of 68 nM. The maximum block observed in the mDRG assay was 89% at the highest tested concentration (300 nM), while the corresponding data for unconjugated peptide 1 was 96% at the highest tested concentration (300 nM).

Figure 4. hNaV inhibition by conjugate 5 in HEK293 cells. a) Manual whole cell patch clamp recordings show block of human NaV1.7 sodium currents. TTX is the positive control. b) Doseresponse curve. IC50 is 297±91 nM (n=5). c). NaV isoform selectivity and manual patch clamp data for conjugate 5 with peptide 1 for comparison, in nM, mean±SEM, with n≥3. n.d. = not determined.

Mouse Pharmacokinetics Pharmacokinetic studies were conducted to discern if the increased molecular weight and superior FcRn-mediated in vivo circulating half-life of immunoglobulin proteins led to HLE for GpTx-1 peptides after conjugation (Figure 5). Conjugate 5 was administered subcutaneously to mice and the serum concentration-time profile was evaluated using ligand binding mass


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spectrometry techniques (LBMS).25 Intact conjugate concentrations were calculated from quantitation of a peptide fragment from peptide 1 by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Total antibody concentrations were measured via a peptide fragment from the Fc domain of the carrier and correspond to the sum of both intact conjugate and remaining antibody fragment after any warhead cleavage in vivo. The intact conjugate was detectable in serum seven days post dosing, with a half-life of 80 h (Table S2). The maximal concentration was reached at 24 h. Analysis of intact conjugate vs. total antibody by LBMS showed minimal degradation until day 7 with an intact:total ratio of 0.62, highlighting the proteolytic stability of GpTx-1 peptides in vivo when renal filtration is minimized by antibody conjugation (Figure S10). In contrast, a related but non-conjugated GpTx-1 peptide [Ala5]GpTx-1, compound 22,11 dosed in mice subcutaneously was rapidly cleared, with a halflife of 0.6 h as anticipated for a small ~4 kDa peptide. Thus, conjugation to an antibody with a PEG linker increased the half-life by approximately 130-fold.

Figure 5. Pharmacokinetic study in male CD-1 mice (n = 3/time point). Conjugate 5 was dosed subcutaneously at 10 mg/kg and serum samples of intact conjugate were monitored by ligand


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binding mass spectrometry.25 Peptide 22 was dosed subcutaneously at 5 mg/kg in a separate cohort and monitored by LC-MS/MS. Data are mean ±SEM for each time point.

Biodistribution of peptide-antibody conjugate to nerves A criterion for conjugates to affect desired therapeutic end points is distribution to sites of target expression. To this end, we explored NaV1.7 inhibitory peptide-antibody conjugate access to peripheral nerve elements where NaV1.7 is localized. We dosed E384C conjugate 5 and the parent antibody 3 in wild-type CD-1 mice intravenously at 20 mg/kg and collected dorsal root and sciatic nerve at 1 and 7 days post-dosing. We detected the antibody backbone using an Alexa488-labeled goat anti-human IgG and visualized signals with fluorescence microscopy. Conjugate 5 biodistributed to nerve elements to a much greater extent than parent antibody 3 at either 1 or 7 days post-dosing, suggesting that nerve access or retention was dependent on the NaV1.7 inhibitory peptide component of the conjugate (Figure 6). The plasma concentrations of conjugate 5 on day 1 and day 7 were 185 and 30 nM, respectively. To determine if nerve biodistribution was dependent on the presence of NaV1.7 protein, we evaluated conjugate 5 access to and co-localization with nerve fiber markers in WT and NaV1.7 KO mice. Conjugate 5 accumulated in dorsal roots (Figure 7) and sciatic nerves (Figure S11) in WT mice to a much greater extent than in NaV1.7 KO mice 7 days post-dosing. The plasma concentration of conjugate 5 in KO mice on day 7 was 17 nM, within 2-fold of the plasma concentration in WT mice at this time point. In addition, in WT mice conjugate 5 co-localized with both the nerve fiber marker PGP9.5 and the C-fiber marker peripherin, both of which have been reported to be present along NaV1.7-positive nerve fibers,26 indicating that nerve access or retention was dependent on NaV1.7 protein expression. Conjugate 5 also biodistributed to olfactory sensory


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neurons, another site of NaV1.7 expression,6 to a greater extent in WT but not NaV1.7 KO mice (data not shown). Taken together, these data indicate that conjugate 5 biodistribution to nerve elements is dependent on both NaV1.7 inhibitory peptide and NaV1.7 protein.

Figure 6. Biodistribution of NaV1.7 peptide-Ab conjugate to mouse dorsal root (DR) and sciatic nerve (SN). Immunoreactivity of conjugate 5 at day 1 (D1) post-injection in DR (a) and SN (c), compared to antibody 3 in DR (b) and SN (d). Immunoreactivity of conjugate 5 at day 7 (D7)


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post-injection in DR (e) and SN (g), compared to antibody 3 in DR (f) and sciatic nerve (h). Conjugate 5 is detected in nerve elements to a greater extent than parent antibody 3. Green denotes conjugate 5 or antibody 3 staining, and blue denotes cell nuclei staining in sagittal sections. Scale bars are 50 microns.

Figure 7. NaV1.7 peptide-antibody conjugate labels neuronal processes. Conjugate 5 immunoreactivity co-localizes with the neuronal marker PGP9.5 in DR (a-c) of WT but not DR (d-f) of NaV1.7 KO mice. Conjugate 5 immunoreactivity co-localizes with peripherin, a C-fiber marker, in DR (g-i) of WT but not DR (j-l) of NaV1.7 KO mice. Left panels denote conjugate 5 staining in green, middle panels denote PGP9.5 or peripherin staining in red, and right panels overlay green and red signals with yellow color indicating co-localization in saggital sections. Scale bars are 50 microns.


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Discussion Ion channel inhibitory peptides isolated from the venom of piosonous species have potential for altering activity of channels associated with human disorders. We have previously shown that synthetic analogs of the NaV1.7 inhibitory peptide GpTx-1 demonstrated increased selectivity against other NaV channels. Given the lack of progress with generation of NaV1.7 blocking antibodies,10 we investigated a FcRn-based delivery strategy with conjugation of GpTx1 peptides to a carrier antibody to investigate an alternate large molecule approach for ionchannel inhibition. Ion-channel inhibitory peptides can be genetically fused to carrier proteins including Fc domain of antibodies and human serum albumin.27 We focused on site-specific chemical conjugation to cysteine engineered antibody carriers. Site-specific conjugation methods limit heterogeneous mixtures with respect to both drug attachment site and DARs observed under nonspecific conjugation conditions.19 Introduction of specific cysteines allows exploration of multiple conjugation sites on the antibody scaffold, while genetic fusion methods limit warhead attachment to either termini of the carrier protein. Conjugation to internal sites can confer pharmacokinetic and pharmacodynamic benefits.21,22 GpTx-1 peptides contain a native Cterminal carboxamide that is required for potent NaV1.7 block,12 and the introduction of this modification by recombinant methods is challenging. Using recombinant expression in HEK293 cells, studies of NaV1.7 inhibitory peptide huwentoxin-IV focused on less potent C-terminal carboxylic acid-containing peptide analogs.28 In solid phase peptide synthesis (SPPS), selection of the appropriate solid support produces the desired C-terminus directly upon cleavage from the resin.11,29 Furthermore, we identified potent analogs with unnatural amino acids in the peptide sequence, e.g., 1-naphthylalanine.12 Thus, we sought a derivatization method that would allow inclusion of unnatural amino acids. Other complexities with genetic fusion methods include


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proteolytic cleavage of expressed peptidic linkers connecting the bioactive warhead to carrier protein30 and potential difficulty of correctly folding disulfide-rich peptides when expressed as fusion proteins in heterologous cells.31 Misfolding of cysteine-rich peptides can increase upon modification of the WT sequence and can lead to mixtures of disulfide isomers with reduced bioactivity (data not shown). The desired isomer can be purified at the peptide stage after SPPS using standard reversed-phase chromatography, unlike potentially challenging separations of peptide folding isomer-antibody mixtures from recombinant expression. Therefore, we selected a site-specific chemical conjugation strategy to attach purified bioactive GpTx-1 peptides to a carrier antibody. The design of NaV1.7 inhibitory peptide-antibody conjugates differs in several aspects from the well-known class of hybrids known as antibody-drug conjugates or ADCs.32 First, the primary purpose of conjugating an antibody to a cytotoxic payload is to target an ADC to cancer cells expressing the antigen. Second, ADCs are typically designed to be internalized by the targeted cell. This event can trigger, via a cleavable linker, release of the cytotoxic agent into the intracellular space. In our case, we selected a non-targeting antibody as the carrier for the peptide warhead and incorporated a non-cleavable PEG linker between the peptide and the antibody to block NaV1.7 on the cell surface with intact conjugate. The conjugation chemistry protocol was optimized with consideration to unwanted sidereactions in order to deliver conjugates in high yields. Linker attachment chemistry was chosen to be orthogonal to peptidic side-chain functionalities and tolerant of the disulfide-rich peptide. Cysteine-engineered antibodies are produced in cells capped as disulfides with endogenous thiols and conjugation must be preceded by their removal. We tested both partial reduction which selectively reduces the caps as well as a redox protocol which uses excess reductant and


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subsequent reformation of any cleaved intra-chain disulfides via mild oxidant to reduce the engineered cysteines. It was interesting to note the variation in alkylation proficiency across the multiple cysteine mutant sites designed with similar in silico parameters. Reactivity screening allowed us to prioritize the list of mutants that can be preferentially employed to generate conjugates for future applications. Optimal conversions were obtained using redox conditions, and peptide mapping validated the fidelity of peptide attachment site on the antibody. Conjugates exhibited a loss in potency compared to the naked peptide, potentially due to steric effects limiting peptide access to NaV1.7. The sodium channel selectivity profile of the parent peptide was maintained upon conjugation. SAR efforts revealed approaches for improving potency by increasing peptide loading on the conjugate. Heterogeneous high DAR ADCs prepared by non-selective methods display divergent potency and pharmacokinetic attributes.33 Further studies are warranted to explore the potential of novel homogenous higher DAR peptide-antibody conjugates like 11 prepared by a site-selective method employing a dimeric linker to increase warhead loading. Variation in site of peptide-linker attachment also affected potency over a 10-fold range, and suggested increased access to the target channel on the cell surface in vitro with peptide linked to cysteine sites with higher potency like D88C, E99C and T487C. Detailed characterization of higher potency constructs will be the subject of future publications. Mouse pharmacokinetic studies demonstrated HLE of the active peptide by 130-fold through attachment to the carrier antibody via a stable PEG linker. HLE was achieved by limiting renal filtration through increasing the molecular weight to 157 kDa and potentially from FcRn-based recycling processes.16 Successful HLE of NaV1.7 inhibitory peptides using a conjugation approach may offer target coverage over less frequent dosing schedules.


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Access of large molecules to nerve fibers in vivo could be limited by the blood nerve barrier comprised of the perineurium, a multicellular layer of epithelioid fibroblast cells with tight junctions that surround nerve fascicles, as well as endoneurial blood vessels.34,35 Biodistribution of NaV1.7 peptide-antibody conjugates to nerve fibers therefore necessitates transcellular or paracellular passage through perineurial and/or endoneurial membranes. Previous ex vivo work demonstrated that the NaV1.7 inhibitory peptide ProTx-II did not impact action potential firing unless the saphenous nerve was first desheathed to enable peptide access,36 highlighting a challenge for effectively delivering naked peptides to drug targets in the nerve. Access of the conjugate to nerve fibers following intravenous dosing could be mediated by FcRn-mediated transcytosis across perineurial and/or endothelial cells. Alternatively, some conjugate could extravasate from highly vascularized DRG,37 which lack a robust blood nerve barrier,38 and diffuse to proximal dorsal root and distal sciatic nerve regions. In our imaging experiments, conjugate biodistribution to nerve fibers was dependent on both NaV1.7 inhibitory peptide and NaV1.7 ion channel presence, suggesting direct interaction of the peptide moiety with the NaV1.7 target. The conjugate was detected seven days after a single injection, indicating retention in the local nerve microenvironment. Conjugate plasma concentrations fell 6-fold over 7 days and were similar between KO and WT mice on day 7. Since tissue concentration was not measured, target coverage at the nerve is not known. Our qualitative studies do not elucidate the precise level of receptor occupancy by conjugates in nerve fibers. Conjugate 5 plasma concentrations were only 2-3 fold higher than mTTX-S DRG IC50 on day 1, which may explain inability to impact scratching behavior in a mouse histamine-induced pruritis model (data not shown). Previously, we reported that plasma exposures well over in vitro IC50 values are required to engage NaV1.7-dependent pharmacodynamic endpoints. 39,40


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Conclusion Identifying large molecule blockers of NaV1.7 has been challenging with no reproducible inhibitory antibodies reported to date.9,10 Our study offers a design template for NaV1.7 blockade using a modality encompassing a NaV1.7 inhibitory peptide coupled to a carrier antibody. The detailed chemistry protocol optimization reported herein can be applied towards HLE and tissue distribution of diverse bioactive peptides. Our results demonstrate that conjugation leads to both peptide HLE and biodistribution to nerve fibers, raising the prospect of inhibiting hyperactive sodium channel function in chronic pain states for a prolonged duration. Design strategies were identified for improved block of NaV1.7 function offering further opportunities for potency optimization.

ASSOCIATED CONTENT Acknowledgements M. Wanska is acknowledged for synthetic assistance and K. Andrews and R. Foti for help with figures and data analysis. Supporting Information Supporting Information is available free of charge via the internet at http://pubs.acs.org Full details of conjugate synthesis and characterization including peptide mapping, pharmacokinetics and biosdistribution imaging study methods and control imaging experiments. References 1. Holmes, D. (2016) The pain drain. Nature 535, S2–S3.


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Competing financial interests All authors are current or former employees of Amgen Inc.


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for table of contents only 79x40mm (300 x 300 DPI)


Engineering Antibody Reactivity for Efficient Derivatization to Generate

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