Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and

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Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation to Enhance Pharmacokinetics Justin K. Murray, Bin Wu, Christopher M. Tegley, Thomas E. Nixey, James R. Falsey, Brad Herberich, Li Yin, Kelvin Sham, Jason Long, Jennifer Aral, Yuan Cheng, Chawita Netirojjanakul, Liz Doherty, Charles Glaus, Tayo Ikotun, Hongyan Li, Linh Tran, Marcus Soto, Hossein Salimi-Mossavi, Joseph Ligutti, Shanti Amagasu, Kristin L. Andrews, Xuhai Be, Min-Hwa Jasmine Lin, Robert S. Foti, Christopher P. Ilch, Beth Youngblood, Thomas J. Kornecook, Margaret Karow, Kenneth W. Walker, Bryan D. Moyer, Kaustav Biswas, and Les P Miranda ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00183 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation to Enhance Pharmacokinetics Justin K. Murray,†,* Bin Wu,† Christopher M. Tegley,† Thomas E. Nixey,† James R. Falsey,† Brad Herberich,† Li Yin,† Kelvin Sham,† Jason Long,† Jennifer Aral,† Yuan Cheng,† Chawita Netirojjanakul,† Liz Doherty,† Charles Glaus,† Tayo Ikotun,† Hongyan Li,◊ Linh Tran,◊ Marcus Soto,◊ Hossein Salimi-Moosavi,◊ Joseph Ligutti,‡ Shanti Amagasu,‡ Kristin L. Andrews,# Xuhai Be,§ Min-Hwa Jasmine Lin,§ Robert S. Foti,§ Christopher P. Ilch,∆ Beth Youngblood,‡ Thomas J. Kornecook,‡ Margaret Karow,† Kenneth W. Walker,† Bryan D. Moyer,‡ Kaustav Biswas,†,^ and Les P. Miranda† †Therapeutic

Discovery, ‡Neuroscience, and ◊Pharmacokinetics and Drug Metabolism,

Amgen Research, One Amgen Center Drive, Thousand Oaks, CA 91320, USA and #Therapeutic

Discovery, ∆Neuroscience, and §Pharmacokinetics and Drug Metabolism,

Amgen Research, 360 Binney Street, Cambridge, MA 02142, USA ^Present

address: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA

02115, USA *To

whom correspondence should be addressed: Dr. Justin Murray Amgen Inc. One Amgen Center Drive MS 29-M-B Thousand Oaks, CA 91320 Phone: 805-313-5640 [email protected] 1 ACS Paragon Plus Environment

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Keywords: Voltage-gated sodium channel, NaV1.7, peptide antagonist, toxin, disulfiderich peptide, antibody conjugate, pharmacokinetics, pharmacodynamics

ABSTRACT Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need for chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptideantibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analoging. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in linker, peptide loading, and antibody attachment site. We found conjugates with 100x improved in vitro potency relative to complementary GpTx-1 analogs, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold whilst retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analog with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss in peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound

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with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

INTRODUCTION The voltage-gated sodium channel NaV1.7 is a well-described target with strong human genetic validation for pain.1,2,3,4 Humans with mutations in the SCN9A gene that encodes the NaV1.7 protein present with different phenotypes at the extreme ends of the pain spectrum. While individuals with a gain-of-function NaV1.7 mutation experience severe pain (paroxysmal extreme pain disorder and primary erythromelalgia),5,6,7,8 those with a loss-of-function NaV1.7 mutation are incapable of pain sensation (congenital insensitivity to pain).9,10 This clinical evidence has sparked and sustained efforts across academic and industrial laboratories to identify a range of entities, including small molecules, peptides and antibodies, capable of potently and selectively blocking NaV1.7 with the goal of developing novel pain therapeutics and mitigating the opioid epidemic in the United States.11,12,13 Here we report recent results with NaV1.7 inhibitory peptideantibody conjugates toward this objective. In one of our research programs we decided to pursue a peptide-antibody hybrid design goal for the identification of a large molecule NaV1.7 inhibitor with altered biodistribution and pharmacokinetics (PK) due to neonatal Fc receptor (FcRn) recycling mechanisms.14 Although a NaV1.7 blocking antibody has been reported,15 its activity has not been reproducible in subsequent batches from hybridomas or by recombinant production.16,17 Given the absence of a bona fide functional or NaV1.7 binding antibody, we decided to prepare NaV1.7 inhibitory peptide conjugates with antibody scaffolds possessing no affinity or functional activity to NaV1.7.


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Naturally occurring peptides that antagonize NaV1.7 with low nanomolar or high picomolar activity have been identified in the venom of tarantula spiders, and progress toward engineering NaV sub-type selective peptide analogs has been achieved.18,19,20 However, the optimization of these peptides for sustained exposure at the site of action in vivo has not yet been addressed. Although the compact, folded structure of disulfide-rich peptides often protects them from proteolysis,21 they suffer from a short in vivo half-life due to rapid renal filtration.22 Several half-life extension strategies exist including PEGylation, albumin binding, protein fusion, etc.23 Conjugation of an antibody to the peptide may confer the most significant improvements due to its large hydrodynamic radius, which decreases renal filtration, and its interaction with FcRn that results in antibody recycling.14 Our efforts to prepare and characterize these peptide-antibody hybrid molecules, related in design but distinct to antibody-drug conjugates (ADCs),24 indicate that the overall properties of a peptide-antibody conjugate are heavily dependent on the attributes and physical properties of the peptide warhead. The structural components of a peptide-antibody conjugate can be broken into three constituents, the peptide, the linker, and the antibody, that can be independently and collectively modified. Two series of NaV1.7 inhibitory toxin peptides, first GpTx-1 and more recently JzTx-V, have been investigated in our laboratory.25,26 Both peptides were discovered via high throughput electrophysiology screening of fractionated tarantula venoms for functional inhibition of NaV1.7 followed by deconvolution of the peptide sequences.27,28,29 Their selectivity against key off-target isoforms NaV1.4 and NaV1.5, expressed in skeletal muscle and cardiac tissue respectively, was significantly improved through multi-attribute positional scan analoging.30,31,32 This approach used an integrated 4 ACS Paragon Plus Environment


and automated peptide preparation process to synthesize, oxidatively fold, and purify several series of single substitution analogs (i.e. Ala, Glu, Lys, Arg, Trp and 1naphthylalanine scans).33,34,35 Screening these compounds elucidated the distinct positional physiochemical attributes of the two toxin peptide series, clearly identifying residues on the putative NaV1.7 binding face as well as solvent-exposed amino acid positions. NMR and x-ray crystal structures of the peptides facilitated structure-activity relationship (SAR) studies and contributed to the recent optimization of JzTx-V analogs through substitution of unnatural amino acids at key positions for improving potency, in particular 5-bromotryptophan for Trp24.36 Suitable sites for attachment of the linker to JzTx-V with minimal loss of activity were also determined, and incorporation of a propargylglycine (Pra) at one of these positions enabled copper-catalyzed 1,3-dipolar Huisgen cycloaddition or "click" reaction with the azide functionality at one end of a bifunctional spacer to form a triazole linkage.37,38 A reactive bromoacetamide moiety at the distal end of the linker can then be used to conjugate the peptide warhead to a cysteine-derived free thiol in the antibody through formation of a stable thioether bond.39 Our initial effort at antibody conjugation with GpTx-1 peptides served to develop the chemistry and identify architectural features that impacted conjugate preparation and potency.39 The desirable properties for a surface-exposed conjugation site include high expression of the antibody with the engineered cysteine, high reactivity of the reduced thiol with the incoming electrophile, low propensity for intermolecular dimerization, stability of the linkage to the peptide, and minimal reduction in potency of the warhead after conjugation. We site specifically conjugated GpTx-1 analogs to engineered cysteine residues at a variety of surface-exposed positions on a carrier antibody using different 5 ACS Paragon Plus Environment

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polyethylene glycol linkers with retention of NaV1.7 inhibitory activity.40,41,42,43,44 Potency of the peptide-antibody conjugates was dependent on the conjugation site within the antibody, length of the linker, and loading of the peptide. The most potent compound with an in vitro hNaV1.7 IC50 value of 57 nM was a construct comprising a divalent linker with a total of four copies of a GpTx-1 peptide per antibody (peptide drug to antibody ratio of 4 or DAR4).39 The mouse PK profile of a DAR2 conjugate was investigated and found to have an extended in vivo half-life relative to a naked GpTx-1 peptide analog (130x). Imaging studies showed the potential of this hybrid modality to biodistribute to NaV1.7-expressing nerve fibers. However, the lead compound in this series of GpTx-1 peptide-antibody conjugates, 1 (Table 1), was 30-fold less potent (NaV1.7 IC50 = 250 nM) than the component naked peptide, had low in vivo target coverage as judged by the ratio of the serum concentration to the in vitro IC50, and no activity in a mouse histamine-induced pruritis model. The need to increase activity of the conjugate led us to expand our natural peptide collection with additional tarantula venoms, the screening of which resulted in identification of the more potent NaV1.7 inhibitory peptide warhead JzTx-V. Further optimization for selectivity and derivatization yielded the analog AM-0422, CyA-[Nle6;Pra17;Glu28]JzTx-V(1-29) (NaV1.7 IC50 = 0.8 nM, NaV1.4 IC50 = 103 nM, and NaV1.5 IC50 = 966 nM), a peptide warhead that is 10-fold more potent than the previous GpTx-1 peptide analog26 and a starting point for the conjugation studies reported here.



RESULTS AND DISCUSSION Identification of Potent Peptide-Antibody Conjugates. We first sought to generate a compound with increased in vivo target coverage by improving the in vitro activity of the conjugate. An initial series of NaV1.7 inhibitory peptide-antibody conjugates was prepared from the potent and selective JzTx-V peptide analog AM-0422 and a nonNaV1.7 targeting, anti-2,4-dinitrophenol (αDNP) human IgG1 antibody with a cysteine mutation at a preferred surface-exposed residue, E384C, by varying the loading and nature of the polyethylene glycol (PEG) linker. The “symmetric” DAR2 conjugate 2 with a linear PEG11 linker (Figure 1A) had a hNaV1.7 IC50 value of 74 nM in a whole cell patch clamp electrophysiology assay on the automated PatchXpress® (PX) system in heterologous HEK293 cells,25 a 3x increase in potency relative to GpTx-1 conjugate 1, reflecting the direct impact of the JzTx-V peptide warhead. However, the peptide-Ab conjugate was 90-fold less potent than the unconjugated peptide and 13-fold less potent than the peptide-linker conjugate (hNaV1.7 IC50 = 5.6 nM), which suggested potential for architectural optimization to improve potency. To initiate this effort, we explored the structural engineering concepts of linker configuration and peptide loading.37,39 Presentation of the peptide was altered in “asymmetric” DAR2 conjugate 3 by attaching a dimeric peptide-PEG23 linker to only one of the available engineered cysteines (Figure 1B). JzTx-V conjugate 3 was 9-fold more potent (hNaV1.7 IC50 = 7.8 nM) than “symmetric” conjugate 2 (Table 1). It is possible that the increased linker length or asymmetric geometry may improve in vitro potency by facilitating the natural propensity of the warhead to interact with the cell membrane, a key attribute of NaSpTx Family 3 toxin peptides like JzTx-V and ProTx-II that differentiates them from GpTx-1, HwTx-IV,


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and other members of NaSpTx Family 1.45,46 As a next step, the peptide loading was increased to DAR4 in conjugate 4 by attachment of a dimeric peptide-linker to each heavy chain (Figure 1C), which led to a concomitant 3-fold increase in potency to a 2.3 nM hNaV1.7 IC50, nearly 25-fold more active than the most potent conjugate in the GpTx-1 series (also DAR4). Therefore, structure modifications with variations in peptide warhead presentation or loading identified NaV1.7 inhibitory peptide-Ab conjugates with single digit nanomolar in vitro potency. The compounds were then examined in mouse PK studies for further characterization of their in vivo properties.

Figure 1. Models of peptide-αDNP antibody conjugate architectures with different linkers and peptide loading. The attachment point in the antibody at the engineered cysteine is displayed in CPK. A) Symmetric divalent conjugate 2 with PEG11 linker at E384C. B) Asymmetric divalent conjugate 3 with dimeric PEG23 linker at E384C. C) Tetravalent conjugate 4 at E384C. D) Symmetric divalent conjugate 5 with PEG11 linker 8 ACS Paragon Plus Environment


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at T487C. E) Symmetric divalent conjugate 6 with PEG11 linker at D88C. F) Symmetric divalent conjugate 10 with GGS linker at D88C. Heavy chains are displayed in magenta and orange, light chains are displayed in yellow and cyan, and peptides are displayed in green. Table 1. NaV1.7 Inhibitory Peptide-αDNP Antibody Conjugates and in Vitro Activity Conjugate

hNaV1.7

DAR

PX IC50

Antibody

Linker

Peptide

Site

Peptide

Peptide

Charge

hNaV1.7 PX IC50

(nM)†

(nM) 1

250

2

E384C

PEG11

AM-1050‡

+6

8.0

2

75

2

E384C

PEG11

AM-0422

+6

0.8

3

7.8

2

E384C

Bis-PEG23

AM-0422

+6

0.8

4

2.3

4

E384C

Bis-PEG23

AM-0422

+6

0.8

5

2.8

2

T487C

PEG11

AM-0422

+6

0.8

6

3.3

2

D88C

PEG11

AM-0422

+6

0.8

7

10.4

2

D88C

PEG11

AM-1647

+3

1.0

8

3.1

2

D88C

PEG11

AM-5695

+4

0.3

9

16.4

2

D88C

PEG11

AM-6122

+2

1.5

10

1.6

2

D88C

GGS

AM-2752

+2

0.3

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 = 10 different cells, with two to three data points per concentration to fit each IC50; See Supporting Information for hNaV1.4, hNaV1.5, mouse NaV1.7, and rat NaV1.7 IC50 values for select conjugates. ‡ [Ala5,Phe6,Pra13,Leu26,Arg28]GpTx-1(1-34) in ref. 39 †

Pharmacokinetics and Bioimaging in Mice. Conjugates 2–4 were dosed in mice at 5 mg/kg body weight and total levels of antibody were measured to ascertain the extent of 9 ACS Paragon Plus Environment

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in vivo half-life extension afforded to the JzTx-V peptide by conjugation to the carrier αDNP E384C antibody. Interestingly, the serum exposures and half-lives of the peptideAb conjugates were much less than those of the parent antibody and the antibody-linker controls (Figure 2; Table S3). In particular, the serum concentrations were lower than the unconjugated controls at the initial 0.5 hr time point and declined rapidly during the first 24 hrs, indicating rapid tissue distribution or degradation of the conjugates. It was observed that increasing peptide accessibility via longer linker length and/or copy number to improve conjugate activity correspondingly decreased the serum exposure, in agreement with the seminal work of Hamblett et al. on drug loading in ADCs.47 All conjugates achieved only a 1x exposure multiple (serum concentration/hNaV1.7 IC50) for ~8 hrs, similar to previous observation with GpTx-1 conjugate 1.39 The increased potency of the JzTx-V peptide-antibody conjugates was seemingly negated by inferior PK properties. This required further investigation and resolution as in vivo activity for small molecule and peptide antagonists of NaV1.7 has required large exposure multiples over the NaV1.7 in vitro IC50.36,48 Serum Concentration (M)

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


1

DNP Ab E384C-PEG

0.1

DNP Ab E384C Conjugate 2

0.01

Conjugate 3 Conjugate 4

0.001 0.0001 0

24

48

72

96

120

144

168

Time Post Dose (hr)



Figure 2. Pharmacokinetic study in male CD-1 mice (n = 3/time point). Conjugates and controls were dosed intravenously at 5 mg/kg body weight, and total concentrations in serum were measured by ligand binding mass spectrometry. Data are mean ± SEM for each time point. αDNP Ab E384C-PEG is the parental antibody with two bis-PEG23 linkers attached similarly to conjugate 4 but without the peptide. The corresponding hNaV1.7 IC50 values for each peptide-Ab conjugate are shown as dotted lines.

To better understand the global biodistribution of the conjugates, control conjugate 1 and DAR4 conjugate 4 were nonspecifically derivatized with the 64Cu chelator NODA-GA (Figure 3A), tested in vitro (hNaV1.7 IC50 values of 426 and 4 nM, respectively), dosed in mice, and imaged at 20 hr post-dose using whole-body positron emission tomography (PET). The parent antibody largely remained in circulation (Figure 3B), and conjugate 1 had a comparatively reduced serum concentration (3-fold) in good agreement with the results of the previously reported PK experiment.39 Notably, conjugate 4 was found heavily deposited in the liver, with 4-fold higher accumulation compared to parental antibody and conjugate 1, and was localized to a much lesser extent in the kidney and spleen. This was corroborated with an ELISA to quantitate exposure in harvested tissues on unlabeled conjugate 4 (see Supporting Information). While distribution to the target tissue of the peripheral neurons is desired to impact NaV1.7 function in vivo and has been observed with GpTx-1 peptide-Ab conjugates using fluorescence-based imaging techniques,39 it was hypothesized that derivatizing the carrier antibody with increasing copies of the JzTx-V peptide AM-0422 was promoting the aberrant tissue binding and sequestration in the liver. The strategy of improving


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conjugate potency via increased warhead loading was therefore deprioritized and alternate structural engineering was undertaken with the expectation that only two copies of the NaV1.7-inhibitory peptide would be incorporated into future designs. Also, the reduced PK profile observed with conjugate 3 suggested that incorporation of the long, divalent linker was not a viable approach to increasing potency and in vivo target coverage.

A

O

HO

O

O

N O

N RS S

S SR'

NODA-GA-NHS 50 mM sodium phosphate, 2 mM EDTA, pH 7.5

RS S

O

HO

N O

N S SR'

O

O

NH

OH

N

O

HO

N O

N

OH O

HO NODA-GA-NHS

DNP E384C

NODA-GA

NODA-GA

S SR' RS S

1) 3 eq. TCEP 2) 4.5 eq. dhAA 3) NaV1.7 peptide

Peptide HN

O

S S

O

NH Peptide



Figure 3. (A) Reaction scheme for NODA-GA labeled peptide-Ab conjugate synthesis. (B) PET/CT imaging of 64Cu labeled NaV1.7 peptide-antibody conjugates. Conjugates and control were dosed intravenously at 30-40 mg/kg body weight, and subjects were imaged 20 hours post-injection. H = heart, K = Kidney, and L = Liver. Antibody Conjugation Site. Having explored linker configuration and peptide loading without significant improvement in exposure multiples, the potential to increase NaV1.7 inhibitory peptide-antibody conjugate potency through variation of the attachment point to the antibody was investigated using the PEG11 linker. We previously screened a large panel of newly engineered cysteine residues in the αDNP IgG1 scaffold for derivatization with a GpTx-1 peptide and identified conjugation sites that conferred increased NaV1.7 13 ACS Paragon Plus Environment

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inhibitory potency when compared to E384C, e.g., T487C on the heavy chain and D88C on the light chain.39 Applying this design principle to a more potent warhead, the JzTx-V peptide AM-0422 with a PEG11 linker was conjugated to αDNP antibodies with T487C and D88C mutations to produce DAR2 conjugates 5 and 6, respectively (Figures 1D and 1E). It was interesting to see that conjugate 5 (T487C, hNaV1.7 IC50 = 2.8 nM) and conjugate 6 (D88C, hNaV1.7 IC50 = 3.3 nM) demonstrated 20-fold improvements in potency over the corresponding E384C conjugate 2, much greater than the 5-fold improvement observed in the corresponding GpTx-1 series. Moreover, the activity of conjugates 5 and 6 was equivalent to that of conjugates 3 and 4 but without the extended linker or higher peptide loading that had negatively affected PK properties. Thus, employing the JzTx-V warhead provided potent conjugate blockers of NaV1.7 when attached at engineered cysteines located proximal to the two termini of the IgG scaffold, suggesting improved access of the peptide to the target ion channel on the cell surface. To characterize potent D88C and T487C conjugates 5 and 6 in vivo, they were evaluated in a mouse PK study and compared to the corresponding E384C analog (Figure 4). Conjugates 5 and 6 exhibited 1.9- and 2.3-fold lower AUC values, respectively, compared to the E384C conjugate 2. It is noteworthy that the chemical composition of all three conjugates were virtually identical except the site of the cysteine antibody mutation, but they exhibit both different on-target potency and PK properties. As mentioned previously, it is likely that the increased activity against NaV1.7 suggests greater access to the peptide warhead at the D88C and T487C sites. This could potentially correlate with the higher clearance of conjugates 5 and 6 in vivo, due to a greater impact of the more “exposed” warhead peptide on the overall physicochemical



properties of the molecule. Nevertheless, these conjugates maintained serum concentrations that were greater than their in vitro IC50 values for nearly 7 days, and the DNP D88C antibody scaffold was adopted for future analoging described below.

Serum Concentration (M)

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

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1

Conjugate 2 0.1

Conjugate 5 Conjugate 6

0.01

0.001 0

24

48

72

96

120

144

168

Time Post Dose (hr)

Figure 4. Pharmacokinetic study in male CD-1 mice (n = 3/time point). Conjugates were dosed intravenously at 5 mg/kg body weight, and total concentrations in serum were measured by ligand binding mass spectrometry. Data are mean ± SEM for each time point. The corresponding hNaV1.7 IC50 values for each peptide-Ab conjugate are shown as dotted lines.

Reduction of Peptide Net Charge. Preliminary systematic optimization of each component produced NaV1.7 inhibitory peptide-antibody conjugates 5 and 6 that were ~100x more potent in vitro than conjugate 1. However, in vivo they had only slightly improved exposure multiples of ~3x at 48 h due to rapid initial clearance. We postulated that a specific molecular attribute of the JzTx-V warhead might potentially impact the performance of the conjugate in vivo and that it might be necessary to revisit the peptide


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SAR from a different perspective. Examination of the peptide sequence reveals that JzTx-V has a high isoelectric point (pI ~ 10.5-11) due to the presence of multiple basic Lys and Arg residues. Attaching two (or four) JzTx-V peptides to the DNP antibody dramatically changes the pI of the resulting conjugate. Shifts in pI ≥ 1 unit have been reported to have significant effects on antibody pharmacokinetics and tissue distribution as witnessed in our initial PK experiments.49,50 Therefore, we hypothesized that lowering the pI of our JzTx-V peptide analogs while retaining NaV1.7 potency and selectivity might improve the in vivo PK properties of their corresponding antibody conjugates and thus increase target exposure. Like many natural toxin peptides, JzTx-V is highly basic. It has a net formal charge of +6, containing a total of seven arginine and lysine residues and a free Nterminus but only one glutamic acid, one aspartic acid, and an amidated C-terminus (Table 2). For simplicity, the peptide net formal charge is considered rather than the pI (see Supporting Information), assuming one positive charge (at neutral pH) for the Nterminal amine and each arginine and lysine residue in the sequence, one negative charge for each glutamate and aspartate residue, and all other residues being neutral. This feature of high positive charge may have evolved to facilitate nonspecific interaction with the negatively charged cell membrane or specific binding to the voltage sensor domain of ion channels. However, inspiration was drawn from analysis of bioactive NaSpTx Family 3 peptide sequences showing net formal charges that range from +6 to -1 (Table 2). Potential strategies for lowering the net charge of JzTx-V while maintaining activity were: 1) substitute one or more of the native basic Arg or Lys residues with a neutral amino acid (e.g. Ala); 2) substitute a native neutral residue with an acidic amino acid



(e.g. Glu); 3) append or insert an acidic residue; and 4) cap the N-terminal free amine. Though lead peptide AM-0422 (net charge = +6) contained the desirable electrostatic substitution of glutamic acid for isoleucine at position 28 to improve selectivity against NaV1.4, it also sacrificed the wild type Glu17 for incorporation of the propargylglycine (Pra) residue used for antibody conjugation. Further analysis of our previous single substitution analoging data (Ala and Glu scans) and docking studies of our JzTx-V NMR structure with a homology model of NaV1.7 described in ref. 36 revealed considerable flexibility at the N-terminus of the peptide. An alternative linker attachment point was identified by substituting Pra for Tyr at position 1, thus restoring the native Glu17 and lowering the net charge. Next it was found that extension of the N-terminus with a Glu residue in combination with Pra1 was also well-tolerated.36 Substitution of Ala14 with Glu, as suggested by the Glu positional scan data26 was combined with the other modifications generating peptide AM-1647, Glu-[Pra1;Nle6;Glu14,28]JzTx-V(1-29), with a +3 formal charge and a hNaV1.7 IC50 value of 1.0 nM. Although the peptide retained activity, the resulting DAR2 αDNP D88C conjugate 7 was 3x less potent than the previous D88C conjugate 6 with a 10.4 nM hNaV1.7 IC50. To regain potency of the peptide-antibody conjugate, the substitution of preferred unnatural amino acids on the hydrophobic putative NaV1.7 binding face of the peptide was incorporated into peptide designs, as described previously.36 Briefly, our docking studies indicated that C-terminal JzTx-V residue Ile29 was pointed toward the lipid bilayer surrounding the channel, and it was found that increasing its length and size to a homophenylalanine (hPhe) residue improved activity. Likewise, Trp24 was modelled to bind in the bottom of the cleft of the NaV1.7 domain II voltage sensor, and substitution


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with 5-bromotryptophan (5-BrW) at this position potentially occupies the pocket more completely, resulting in sub-nanomolar peptide NaV1.7 potencies. (During review of this publication, two structures of ProTx-II in complex with NaV1.7 were reported that directly impact understanding of the peptide-protein interface.51,52) These findings were first combined to produce an analog with a +4 net charge, AM-5695, Glu-[Pra1;Nle6;5BrW24;Glu28;hPhe29]JzTx-V(1-29), which had a hNaV1.7 IC50 value of 0.3 nM as a naked peptide and 3.1 nM as DAR2 αDNP D88C conjugate 8. Further reduction of the net charge of a JzTx-V peptide analog to +2 and below was challenging for two reasons. While additional individual positions that tolerated substitution were highlighted by both natural sequences and our Ala and Glu scanning data (e.g. Ser11Glu, Lys12Ala, Lys12Glu, etc.), the combination analogs were less potent, especially in the peptide-antibody hybrid format. Also, preparation of peptides with lower charge in sufficient isolated yield became increasingly difficult. The oxidative folding of the low net charge analogs suffered from increased precipitation until the order of reagent addition was modified (slow addition of oxidant as final step) and a panel of conditions was screened to identify an optimal co-solvent (10% DMSO) and denaturant (2.0 M guanidine). The DAR2 αDNP D88C conjugate 9 of an optimized +2 net charge analog AM-6122, Glu-[Pra1;Nle6;Ala12;Glu14,28;5-BrW24]JzTx-V(1-29), had a hNaV1.7 IC50 value of 16.4 nM, which was 10-fold less than the 1.5 nM hNaV1.7 IC50 of the peptide alone. To date, conjugates of +1 net charge and neutral JzTx-V peptide analogs have been much less potent (data not shown).



Page 20 of 50

Table 2. JzTx-V Peptide Analogs and NaSpTx Family 3 Peptides with Net Charge45 Position Peptide Name

N-Term -1

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 C-Term

Basic Acidic

Net Charge

AM-0422

NH2

CyA Y C Q K W Nle W T C D

S

K

R

A

C C [Pra] G

L

R

C

K

L

W

C

R

K

E

I

NH2

8

2

6

AM-1647

NH2

E Pra C Q K W Nle W T C D

S

K

R

E

C C

E

G

L

R

C

K

L

W

C

R

K

E

I

NH2

8

5

3

AM-5695

NH2


S

K

R

A

C C

E

G

L

R

C

K

L 5-BrW C

R

K

E

hPhe

NH2

8

4

4

AM-6122

NH2


S

A

R

E

C C

E

G

L

R

C

K

L 5-BrW C

R

K

E

I

NH2

7

5

2

PE Pra C Q K W Nle W T C D

S

E

R

A

C C

C

K

L 5-BrW C

R

K

E

hPhe

NH2

6

4

2

AM-2752 JzTx-V

NH2

Y C Q K W M W T C D

S

K

R

A

E G L R NaSpTx Family 3 C C E G L R

C

K

L

W

C

R

K

I

I

NH2

8

2

6

K-TRTX-Ps1a

NH2

Y C Q K W M W T C D

S

A

R

K

C C

E

G

L

V

C

R

L

W

C

K

K

I

I

NH2

7

2

5

ProTx-II

NH2

Y C Q K W M W T C D

S

E

R

K

C C

E

G

M

V

C

R

L

W

C

K

K

K

L

OH

8

4

4

GsAF 1

NH2

Y C Q K W L W T C D

S

E

R

K

C C

E

D

M

V

C

R

L

W

C

K

K

R

L

NH2

8

4

4

GrTx1 Phrixotoxin-2

NH2

Y C Q K W M W T C D

S

K

R

K

C C

E

D

M

V

C

Q

L

W

C

K

K

R

L

OH

8

4

4

NH2

Y C Q K W M W T C D

E

E

R

K

C C

E

G

L

V

C

R

L

W

C

K

R

I

I

N M

NH2

7

4

3

K-TRTX-Gr2c

NH2

Y C Q K W M W T C D

E

E

R

K

C C

E

G

L

V

C

R

L

W

C

K

K

K

I

E W

OH

8

6

2

K-TRTX-Gr2b

NH2

Y C Q K W M W T C D

E

E

R

K

C C

E

G

L

V

C

R

L

W

C

K

K

K

I

E E G

OH

8

7

1

JzTx-45

NH2

Y C Q K W M W T C D

S

E

R

K

C C

E

G

Y

V

C

E

L

W

C

K

Y

N

L

G

NH2

5

4

1

K-TRTX-Ec2c

NH2

Y C Q F K M W T C D

S

E

R

K

C C

E

D

M

V

C

R

L

W

C

K

L

N

L

OH

6

5

1

K-TRTX-Cj2b

NH2

Y C Q K W M W T C D

S

E

R

K

C C

E

G

Y

V

C

E

L

W

C

K

Y

N

M

OH

5

5

0

K-TRTX-Ec2b

NH2

Y C Q K F

L W T C D

T

E

R

K

C C

E

D

M

V

C

E

L

W

C

K

Y

K

E

OH

6

7

-1

K-TRTX-Ec1a

NH2

Y C Q K F

L W T C D

T

E

R

K

C C

E

D

M

V

C

E

L

W

C

K

L

E

K

OH

6

7

-1

Concensus

NH2

Y C Q K W M W T C D S/T/E E/K R K/A C C

E

D/G L/M V/R C R/E L

W

C K/R K/Y I/K/N

W

G

I/L

Pharmacokinetics of Conjugates with Reduced Net Charge. NaV1.7 inhibitory peptide-antibody conjugates 6–9 were tested in mice to determine if lowering the net charge of the peptide warhead would improve the PK properties of the molecules. Indeed, an inverse correlation was observed with reduction of peptide net charge increasing total exposure of the conjugate (Figure 5). Conjugate 9 with the +2 net charge peptide AM-6122 had an 18-fold higher AUC than conjugate 6 with +6 net charge peptide AM-0422 and was within 2-fold of the parent antibody control. Incorporation of hydrophobic amino acids hPhe29 and 5-BrW24 did not seem to adversely impact conjugate PK. Although conjugate 9 had the highest serum concentration, conjugate 8 with the +4 net charge peptide AM-5695 had the highest exposure multiple because of the 5-fold difference in relative NaV1.7 IC50 values. These in vivo results demonstrate the profound impact of the peptide pI on the corresponding antibody conjugate, the ability to alter those effects through modification of the peptide, and the multi-parameter interplay which needs to be balanced for overall optimization of hybrid profiles.


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


Serum Concentration (M)

Page 21 of 50

1

DNP Ab D88C 0.1

Conjugate 6 Conjugate 7 Conjugate 8

0.01

Conjugate 9 Conjugate 10 0.001 0

24

48

72

96

120

144

168

Time Post Dose (hr)

Figure 5. Pharmacokinetic study in male CD-1 mice (n = 3/time point). Conjugates were dosed intravenously at 5 mg/kg body weight, and total concentrations in serum were measured by ligand binding mass spectrometry. Data are mean ± SEM for each time point. The corresponding hNaV1.7 IC50 values for each peptide-Ab conjugate are shown as dotted lines.

Potency Optimization and Pharmacology of Conjugate Derivatized with a Low Charge Peptide. Since the initial conjugates 7 and 9 with formal peptide charges ≤ 3 in Table 1 had NaV1.7 IC50 values greater than 10 nM, a final round of structural optimization was undertaken to identify a lower charged JzTx-V peptide-antibody conjugate with single digit nM in vitro potency that could be tested in a pharmacodynamic model of NaV1.7 activity. This was accomplished through a change from PEG11 to a peptidic glycine-glycine-serine (GGS) linker (Figure 1F) and an additional analog series of 20 +2 net charge peptide-antibody conjugates (see Supporting Information). Addition of pyroglutamic acid (pGlu) at the N-terminus and substitution of



glutamic acid for the native lysine at position 12 produced AM-2752, pGlu[Pra1;Nle6;Glu12,28;5-BrW24;hPhe29]JzTx-V(1-29), with a +2 formal charge and hNaV1.7 IC50 value of 0.3 nM, which was incorporated into αDNP D88C antibody conjugate 10 with the GGS linker and retained potency within ~5x of the peptide (hNaV1.7 IC50 = 1.6 nM). Although the PK profile of conjugate 10 was not equal to that of our intial +2 peptide conjugate 9 in terms of serum concentration, it did have superior exposure due to its 10-fold increased activity (Figure 5). Conjugate 10 was evaluated in a NaV1.7-dependent pharmacodynamic model in mice that has been previously employed in our labs with small molecule and peptide NaV1.7 antagonists.36,48 In the histamine-induced pruritis model, C57Bl/6 mice are pretreated with compound or the antihistamine diphenhydramine dosed orally at 30 mg/kg as the positive control and are then are subjected to an intradermal histamine challenge with the number of resultant scratching bouts observed over a 30 minute time period. At a 400 mg/kg subcutaneous (s.c.) dose administered 24 hours prior to testing, conjugate 10 had a modest but significant effect in this model, reducing scratching by 50%. This dose had no effect on locomotor activity in an open field activity study in naïve mice (Supporting Information). Analysis of the intact conjugate 10 serum concentration (370 nM) showed a potential 230-fold serum exposure multiple over the human NaV1.7 IC50. However, this concentration is only 34-fold over the relevant mouse activity (mNaV1.7 IC50 = 11 nM), which is much lower than what has been required for in vivo activity with a naked JzTxV peptide (100x) and may explain the lack of robust activity.36,53 Extending the duration of conjugate 10 pre-dosing to 72 h before testing, to potentially facilitate increased biodistribution to the target, was not found to be effective.


Page 22 of 50

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Figure 6. (A) Reduction of scratching bouts in a mouse histamine-induced pruritis model with conjugate 10 (s.c. dosing 24 hr or 72 hr pre-test) with DPH as the positive control (diphenhydramine, 30 mg/kg p.o. dosing 90 min pre-test) in male C57Bl/6 mice, n = 7–10/group. (A) *, p < 0.05; ***, p < 0.001 by one-way ANOVA with Dunnett’s multiple comparison test; (B) Intact serum exposures with calculated multiples using human and mouse NaV1.7 IC50 values.



NaV1.7 inhibitory JzTx-V peptide-antibody conjugates that are 150-fold more potent than previous GpTx-1 lead conjugates have been developed with suitable pharmacokinetic properties to enable serum concentrations in mice that are >200x the human NaV1.7 IC50 values. The challenge throughout has been that increases in conjugate potency have tended to track closely with decreases in serum exposure. These properties diverged slightly through toxin peptide analoging aimed at charge reduction, and our methods and results may be of utility in other endeavors requiring the tuning of peptide physical properties to improve target exposure of their conjugates. Further optimization of αDNP antibody-JzTx-V peptide hybrids with respect to peptide charge and linker has not yet produced a molecule with a more robust response in the mouse histamine-induced pruritis model (see Supporting Information). Full block of this or another PD response may necessitate identification of a peptide warhead with greater activity against mouse NaV1.7, either through screening of additional venom fractions or a focused peptide analoging campaign. Another alternative would be to evaluate conjugates in a NaV1.7-dependent pharmacodynamic model in a different species (e.g. rats) where these conjugates do not exhibit such a large shift in potency relative to human NaV1.7 (see Supporting Information). Even if these strategies were fruitful, the doses used in the pharmacodynamic studies reported herein were impractically high and would have to be reduced by about 100-fold before consideration for further development. Our attempts to develop NaV1.7 inhibitory peptide conjugates for the potential treatment of chronic pain have been driven by the ability of the antibody carrier to extend the in vivo half-life and thereby enable biodistribution of the peptide warhead to the target. NaV1.7 is expressed in peripheral axons, which are protected by the perineurium,


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a multicellular layer of epithelioid fibroblast cells with tight junctions surrounding nerve fascicles, and endoneurial blood vessels that together form the blood-nerve barrier.54 We initially hypothesized that the FcRn interaction common to antibodies might facilitate transcellular passage through perineurial and/or endoneurial membranes and grant NaV1.7 peptide-antibody conjugates access to nerve fibers in vivo and have advantages over other approaches. Disruption of the blood-nerve barrier through co-administration of hypertonic saline was not thought to be suitable for chronic administration.55 Other half-life extension moieties, like high molecular weight PEG, would likely restrict compounds to the vasculature.32 Non-covalent binding to albumin raised concerns around potentially lowered levels of free drug outside the vasculature. Early imaging studies with GpTx-1 peptide-antibody conjugates were encouraging, showing compound accumulation at NaV1.7-expressing neurons.39 However, after significant molecular optimization the high exposure multiples required for small effect sizes in vivo with αDNP antibody-peptide conjugates in this report do not demonstrate a clear advantage in target access for these hybrids. Another potential path forward is to switch from a carrier to a targeting antibody that either binds to NaV1.717 to increase potency56 or facilitates receptor-mediated delivery of the conjugate across the blood-nerve barrier using concepts that have been developed for crossing the blood-brain barrier.57

Conclusion.

Utilization of the JzTx-V peptide warhead with a reduced formal charge

produced a 150-fold increase in NaV1.7 peptide-antibody conjugate potency with an improved pharmacokinetic profile and exposure multiple. However, reaching the required peptide-antibody hybrid levels for NaV1.7 inhibition at the peripheral neuron



Page 26 of 50

behind the blood-nerve barrier to elicit robust PD effects at a reasonable dose has proved challenging. The identification of a long-lived NaV1.7 antagonist has thus far involved the design and development of peptide warheads, antibody scaffolds, linkers, and conjugation chemistries. Several rounds of optimization were needed to balance the interplay of on-target potency and pharmacokinetics properties. Despite significant engineering, our JzTx-V peptide-αDNP antibody conjugates will require at least an additional 10-fold improvement in potency against mouse NaV1.7 (i.e., IC50 < 1 nM) and an exposure multiple of ~100 for robust target engagement by these hybrid molecules in vivo. The strategies developed for optimization of these carrier antibody conjugates could have future utility for the development of bi-specific peptide-antibody hybrids for NaV1.7 and other drug targets.

METHODS Materials. Nα-Fmoc protected amino acids were purchased from Novabiochem (San Diego, CA), Bachem (Torrance, CA), ChemPep (Wellington, FL), or GL Biochem (Shanghai, China). Rink Amide MBHA resin was purchased from Peptides International (Louisville,

KY).

The

following

reagents

were

also

purchased:

N,N-

diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), acetic acid, piperidine, 4methyl piperidine, 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane, oxidized glutathione,

reduced

glutathione,

tris(2-carboxyethyl)phosphine

(TCEP),

urea,

iodoacetamide, acetonitrile (ACN), water, methanol, formic acid and trifluoroacetic acid (Sigma-Aldrich, Milwaukee, WI); dichloromethane (DCM, Mallinckrodt Baker, Inc.); N,N-dimethylformamide (DMF), Dulbecco phosphate buffered saline, 1x (DPBS), HALT


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Protease Inhibitor Cocktail (100x), Streptavidin Dynal M-280 magnetic beads and TPCK-treated trypsin (Thermo Fisher Scientific, Waltham, MA); 6-chloro-1-hydroxy benzotriazole (6-Cl HOBt); HPLC-quality water and acetonitrile (Burdick and Jackson); 1 M Tris-HCl buffer, pH 7.5 and 1.0 M Tris-HCl, pH 8.0 (Teknova). Stable cell lines expressing human (h) voltage-gated sodium (NaV) channels (CHO-hNaV1.3, HEK293hNaV1.4, HEK293-hNaV1.5, HEK293-hNaV1.7, and CHO-hNaV1.8) were used for experiments.

Stable [13C6,15N]-leucine labeled VVSVLTVLHQDWLNGK flanking

peptide was obtained from Mid-West Biotech, Inc. (Fishers, IN). Biotinylated murine anti-human IgG Fc monoclonal antibody (mAb, Clone no. 1.35.1) was prepared and purified as previously described.58

Peptide Synthesis. Rink Amide Chem Matrix resin (0.2 mmol, 0.45 mmol/g loading, 0.444g, Matrix Innovation) was weighed into a CS BIO reaction vessel. The reaction vessel was connected to a channel of the CS BIO 336X automated peptide synthesizer, and the resin was washed 2 x DMF and allowed to swell in DMF for 15 min. Fmocamino acid (1.0 mmol, Midwest Biotech or Novabiochem) was dissolved in 6-chloro-1hydroxybenzotriazole (6-Cl-HOBt, Matrix Innovation) in DMF (0.4 M, 2.5 mL). To the solution was added 1,3-diisopropylcarbodiimide (DIC, Sigma-Aldrich) in DMF (1.0 M, 1.0 mL). The solution was agitated with nitrogen bubbling for 15 min to accomplish preactivation and then added to the resin. The mixture was shaken for 2 h. The resin was filtered and washed with DMF (3 times), DCM (2 times) and DMF (3 times). Fmocremoval was accomplished by treatment with 20% piperidine in DMF (5 mL, 2 x 15 min, Fluka). The resin was filtered and washed with DMF (3 times). All residues were single 26 ACS Paragon Plus Environment


coupled through repetition of the Fmoc-amino acid coupling and Fmoc removal steps described above. Peptide Cleavage. After final Fmoc-removal from the N-terminal residue, resin-bound linear peptide (0.2 mmol scale) was transferred to a 25 mL solid phase extraction (SPE) filter tube, washed with DMF (3 times), DCM (3 times), and dried under vacuum. To the resin was added triisopropylsilane (1.0 mL), 3,6-dioxa-1,8-octane-dithiol (DODT, 1.0 mL), water (1.0 mL), trifluoroacetic acid (TFA, 15 mL), and a stir bar, and the mixture was stirred for 3 h. The mixture was filtered into a 50 mL centrifuge tube. The resin was washed with TFA (~ 5 mL), and the combined filtrate was concentrated by rotary evaporation in Genevac for 2 h. To the residue (~5 mL) was added 40 mL cold diethyl ether and a white precipitate formed. The mixture was centrifuged and the ether was decanted. To the tube was added another 40 mL of cold ether, and the precipitate was stirred. The mixture was centrifuged, and the ether was decanted. The solid was dried under vacuum. Peptide Folding and Purification: In a 1 L PP bottle was prepared a folding buffer with water (800 mL), acetonitrile (100 mL), cysteine solution in water (1 M, 1 mL), and cystine dihydrochloride in water (0.5 M, 6.667 mL). To the crude linear peptide (100 mg) was added 5 mL acetonitrile and 5 mL water. The mixture was vortexed to complete dissolution of the peptide. The peptide solution was added to the buffer followed by TrisHCl pH 8.0 (1M, 100 mL). The pH value was measured to be 8.0. The folding mixture was allowed to stand at 4 °C for 18 to 72 h. A small aliquot was removed, and the sample was analyzed by LC-MS to ensure that the folding was complete. The solution was quenched by the addition of TFA to pH 2.5, and the aqueous solution was filtered.


Page 28 of 50

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The filtered solution (1000 mL, 100 mg peptide) was loaded onto a preparative HPLC column (Phenomenex Synergi 4µm MAX-RP 80A AXIA, 250 x 30 mm) using an Agilent preparative loading pump. The column was attached to a prep HPLC, Agilent/LEAP prep LC-MS, and the peptide was eluted to afford pure folded peptide. The final yields varied with different sequences and generally range from 5-10%. Peptide Purity Characterization. The purity of synthesized compounds (peptides and peptide-linkers) was determined by LC-MS analysis by elution from a Phenomenex MaxRP column (2.5 µm, 2.0 x 50 mm) using a gradient of 5-50% acetonitrile in H2O (0.1%TFA) over 10 min at a flow rate of 750 µL/min on an Agilent 1290 LC-MS system. All peptides for biological testing were ≥95% pure. All peptide-linkers were >95% in purity. Conjugate purity by size exclusion chromatography: listed in Table S1. Peptide-Linker Preparation. Alkyne-containing peptide was subjected to coppercatalyzed 1,3-dipolar cycloaddition with azido-PEG and azido-peptide bromoacetamides of differing lengths to obtain the site-specifically PEGylated peptides with a triazole linkage, thus converting a propargylglycine or Pra residue in the sequence to a 3-(1,2,3triazol-4-yl)alanine or Atz residue. AM-0422 containing a propargylglycine (Pra) at position 17 (48 mg, 7.4 mM in 1.77 mL water), azido-PEG11-bromoacetamide (150 mM, 0.106 mL in water), tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine (TBTA, 10 mM, 0.159 mL in DMSO), sodium ascorbate (50 mM, 1.195 mL in water), and copper (II) sulfate (35 mM, 0.341 mL in water) solutions were prepared fresh. Peptide and reagents were added in the following order to a 50 mL centrifuge tube to achieve the following final concentrations. Peptide stock solution (1.77 mL) was added for a final concentration of 3.7 mM, followed by addition of azido-PEG11-bromoacetamide (0.106



mL) for a final concentration of 4.5 mM. TBTA (0.159 mL) was added for a final concentration of 0.45 mM and mixed thoroughly. Sodium ascorbate (1.195 mL) was then added for a final concentration of 16.9 mM and mixed thoroughly. CuSO4 (0.341 mL) was added for a final concentration of 3.4 mM. The solution was allowed to stand for 1 h at which time it was judged to be complete by LC-MS. To a 25 mL SPE filter tube was added SP Sepharose High Performance resin as a slurry (8 mL) with a pipette. The gel was conditioned with wash buffer (50 mL 20 mM NaOAc, pH = 4.0), loaded with the peptide solution, washed (50 mL, 20 mM NaOAc, pH = 4.0 and 50 mL, 20 mM NaOAc, pH = 4.0, 0.1M NaCl), and eluted with 45 ml 1M NaCl, 20 mM NaOAc, pH = 4.0) into a 50 mL centrifuge tube. The product mixture was purified by loading onto a preparative HPLC column (Phenomenex Luna 5u C18(2) 100A AXIA, 250 x 30mm) at 30 mL/min using an Agilent prep HPLC pump. The column was flushed for 10 min with 10% B solvent. The column was attached to a prep HPLC (Agilent), and the peptide was eluted with a 10-40% B gradient over 60 min (A solvent: 0.1% TFA in water, B solvent: 0.1% TFA in acetonitrile). The fractions were analyzed by LC-MS, pooled, and lyophilized to afford pure peptide-linker construct (AM-0422-PEG11BrAc, 32.34 mg, 56.7% yield) for conjugation to IgG or Fc domain. ESI-MS: Calc. MW for C187H294BrN53O50S6 = 4356.954, Observed MW = 1453.0 (M+3H)3+, 1090.0 (M+4H)4+.

Antibody Preparation. The parent antibody was initially isolated from a XenoMouse® using dinitrophenol (DNP) conjugated to keyhole limpet hemocyanin (KLH) as the antigen. Screening for binders was conducted using DNP conjugated to lysine. The parental antibody was then engineered by site-directed mutation to create surface exposed


Page 30 of 50

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unpaired cysteine residues available for conjugation and cloned into proprietary Amgen mammalian stable expression vectors. The recombinant antibodies were expressed using the Chinese hamster ovary (CHO) host cell line after stable transfection with the vectors created above. Antibodies were purified using a MabSelect Sure column (GE Healthcare, Piscataway NJ) by directly loading the conditioned media and washing the column with Dulbecco’s phosphate buffered saline (PBS). Antibodies were eluted using 100 mM acetic acid pH 3.2 and the elution pools were brought to pH 5.0 using 1 M NaOH. Protein quality was assessed by Coomassie stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high performance liquid chromatography SE-HPLC (BioSep S3000 column, Phenomenex, Torrance, CA, USA) and liquid chromatography electrospray ionization mass-spectrometry.

Antibody Conjugation. DNP mAb (E384C) (kappa/IgG1z) was diluted to ~5mg/mL in reaction buffer (20mM sodium phosphate pH 6.8, 2mM EDTA). Reduction of engineered cysteines was done by incubating mAb with a 3:1 molar ratio of TCEP (4 mM, aq.) to each cysteine at room temperature for 60 minutes. TCEP was removed using a Zeba spin desalting column (40K MWCO). Reduced, desalted antibody was oxidized by incubating with a 4.5:1 molar ratio of dhAA to each cysteine at room temperature for 15 minutes. Lyophilized peptide-linker bromoacetamide (AM-0422-PEG11BrAc) was re-suspended in water at 20 mg/mL immediately prior to conjugation reaction. Peptide and reduced mAb were mixed at a 3:1 molar ratio of peptide to cysteine (6:1 peptide to mAb) in reaction buffer at mAb concentration of 2.5 mg/ml and incubated for 16 h at 20 oC.

The conjugation reaction mixture was subsequently desalted to remove excess free



peptide and loaded onto a HiTrap SP-HP column (GE Healthcare). The conjugate was eluted from the column at a flow rate of 5 mL/min with a gradient of buffer A (100 mM sodium acetate pH 5) and buffer B (100 mM sodium acetate pH 5, 1.2M NaCl) at 020%B over 5 min, 20-50%B over 30 min, 100%B for 5 min, and 0%B re-equilibration for 10 min. The unmodified mAb eluted first, followed by monovalent immunoglobulinpeptide conjugate and then by bivalent immunoglobulin-peptide conjugate 2. Following purification, desired DAR2-containing fractions were combined (~25 mL) and concentrated by centrifugal filtration (Amicon 15, 30K). The solution was buffer exchanged into A5Su storage buffer (10 mM sodium acetate pH 5.0, 9% sucrose) and concentrated to ~10 mg/mL by centrifugal filtration (Amicon 4, 30K). Conjugates were run on reducing SDS-PAGE to confirm conjugation to heavy chain (increase in size by ~5kD) and non-reducing SDS-PAGE to confirm internal disulfides remained intact. LC/MS spectrometry of peptide conjugates was recorded using an Agilent 6224 TOF LC/MS spectrometer with a non-reduced method. After calibration, a 5 µg intact sample was loaded onto a Zorbax 300SB C6 column (1 x 50 mm, 3.5 µm, Agilent Technologies) operation at 75 ºC with a flow rate of 50 µL/min. The conjugate was eluted from the column using a gradient of 90% n-propanol with 0.1% TFA (Buffer B) and 0.1% TFA in water (Buffer A). The gradient condition was 20% to 90 % buffer B in 14 min. From 25.67 mg of DNP E384C mAb and 4.58 mg of peptide-linker construct 12.4 mg of bivalent conjugate 2 was obtained after purification (48% yield).


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Preparation of NODA-GA Labeled Antibody. Anti-DNP mAb (E384C) (kappa/IgG1z), 100 mg at 13.1 mg/mL concentration in A5Su, was diluted to ~5mg/mL in reaction buffer (50 mM sodium phosphate pH 7.5, 2mM EDTA). To the solution was added NODA-GA-NHS (Chematech, 20 equiv., 10 mM in sterile water, 1.360 mL). The tube was capped and shaken (23 °C, 300 RPM) using an Eppendorf Thermomixer R. After 16 h the solution was desalted into A5Su prepared with metal free reagents and analyzed by SEC (97%) and LC/TOF (see Supporting Information). Conjugation to peptide-linker was performed under standard conditions. The low level of NODA-GA loading (1.4 – 1.7 per Ab) did not not significantly impact in vitro activity of the conjugates.

PatchXpress® 7000A Electrophysiology. Adherent HEK293 cells stably expressing NaV1.7 channels were isolated from tissue culture flasks using 1:10 diluted 0.25% trypsin-EDTA treatment for 2-3 minutes and then were incubated in complete culture medium containing 10% fetal bovine serum for at least 15 minutes prior to resuspension in external solution consisting of 70 mM NaCl, 140 mM D-Mannitol, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 with NaOH. Internal solution consisted of 62.5 mM CsCl, 75 mM CsF, 10 HEPES, 5 mM EGTA, 2.5 mM MgCl2, pH 7.25 with CsOH. Cells were voltage clamped using the whole-cell patch clamp configuration at room temperature (~22 °C) at a holding potential of -125 mV with test potentials to -10 mV (hNaV1.4 and hNaV1.7) or -20 mV (hNaV1.5). Test compounds were added and NaV currents were monitored at 0.1 Hz at the appropriate test potential. All compound dilutions contained 0.3% bovine serum albumin to minimize non-specific binding. Cells



were used for additional compound testing if currents recovered to >80% of starting values following compound washout.

At least four different concentrations of test

compound were applied individually, with washout, recovery of current, and resetting of holding voltage between each individual concentration. Percent inhibition as a function of compound concentration was pooled from at least n = 10 different cells, with two to three data points per concentration, and fitting the resulting dataset with a Hill (4parameter logistic) fit in DataXpress 2.0 software to produce a single IC50 curve.59

Pharmacokinetic studies. Animal Welfare. All experimental procedures involving animals were conducted at AAALAC International accredited facilities and were approved by the Amgen Institutional Animal Care and Use Committee. All animals were maintained on a 12:12 h light:dark cycle, with controlled ranges for ambient temperature (68 to 79 °F) and humidity (30 to 70%). Animals were allowed access to food and water ad libitum. Studies were conducted using unblinded protocols. Pharmacokinetic studies with the parental antibody, the parental antibody with a bis-PEG23 linker, αDNP 3A4 D88C antibody and peptide conjugates 2 – 10 were carried out in 6-8 week old male CD-1 mice from Charles River Laboratories. All test articles were dosed via intravenous bolus administration (5 mg/kg) in A5Su buffer (10 mM sodium acetate, 9% sucrose, pH 5.2) via the lateral tail vein. Blood samples were taken at pre-determined time points using a serial sampling scheme with no more than 3 samples taken from an individual mouse. Approximately 50 – 60 µL of blood was collected into a serum separator tube via submandibular vein puncture at each time point 33 ACS Paragon Plus Environment

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and allowed to clot at room temperature for 20 minutes prior to centrifugation (2-8 C for 15 minutes at approximately 11500 x g). The resulting serum was transferred to a 96well plate and frozen at -80 °C until analysis.

Measurement of Test Articles in Serum from Pharmacokinetic Studies. A universal surrogate peptide (VVSVLTVLHQDWLNGK) corresponding to the Fc region of human IgG1 monoclonal antibodies was used for quantitation of total antibody or peptide-antibody conjugates. A stable isotope [13C6,15N]-leucine labeled surrogate peptide VVSV*LTVLHQDW*LNGK was utilized as the internal standard. Calibration standards for each of the test articles (100 – 10,000 ng/mL) together with quality control samples (QC; 300, 750 and 7500 ng/mL) were prepared by spiking a 100 g/mL working stock solution of the appropriate test article in A5Su buffer into mouse serum treated with 2x HALT protease inhibitor cocktail, followed by serial dilution. A 25 L aliquot of each serum sample was added to 25 L of DPBS with 2x HALT protease inhibitor cocktail and 25 L of 50 g/mL biotinylated murine anti-human IgG Fc monoclonal antibody (Clone no. 1.35.1) immobilized on Streptavidin Dynal M-280 magnetic beads in a 96-well plate and vortexed for 1 hour at room temperature to allow for immunocapture treatment. The beads were washed four times with 800 L of 250 mM Tris-HCl buffer (pH 7.5) using a Tomtec Quadra3 instrument (Tomtec, Hamden, CT) with a magnetic nest attachment. Samples were reduced by the addition of 7.5 mM TCEP in denaturation buffer (250 mM Tris-HCl buffer, pH 7.5 containing 8 M urea) with internal standard. Sample alkylation was achieved by adding 25 mM iodoacetamide in water and incubating the samples for 45 minutes at 60 °C. Subsequent digestion was 34 ACS Paragon Plus Environment


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carried out with 0.5 mg/mL trypsin in a microwave set at 400W for 10 min at 30 °C. The digestion was quenched with 20% formic acid. The magnetic beads were pulled down to the sample well by centrifugation at 4500 RPM (~2550 x g) for 10 min and a portion of the supernatant transferred to a fresh plate for LC-MS/MS analysis. A 10 L volume of each supernatant was injected onto an Acquity UHPLC system (Waters, Corp.) and separation of the selected peptides achieved on a Phenomenex Aeris C18 column (2.1x 100mm, 1.7 m) at 70 °C, followed by detection with a Sciex QTRAP® 5500 mass spectrometer (AB Sciex, Foster City, CA) operated in the electrospray positive ion MRM mode. The surrogate peptides and respective ion transitions are listed in Table 3. The mobile phases consisted of (A) 0.1% formic acid in acetonitrile/water (5/95, v/v) and (B) 0.1% formic acid in acetonitrile /water (95/5, v/v). The LC gradient in min/% of mobile phase B was: 0.00/5, 1.00/5, 3.00/35, 3.10/95, 4.60/95, 4.70/5 and 5.00/5. The flow rate was set to 0.5 mL/min and the run time was 5.00 min.

Table 3. Surrogate Peptide Sequences and MRM Ion Transitions. Analyte

Surrogate Peptide

Q1/Q3 MRM

All Test Articles (Total Measurement) Internal Standard for total Ab

VVSVLTVLHQDWLNGK

603.5/805.5

VVSV*LTVLHQDW*LNGK

608.0/812.4

*denotes [13C6,15N]-Leu

Total concentrations of each test article were calculated from calibration curves derived from the peak area ratios (selected peptide/internal standard) using 1/x2 weighted 35 ACS Paragon Plus Environment

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linear least-squares regression. Concentrations and pharmacokinetic analysis were both carried out using Watson LIMSTM software.

Imaging Studies. Radiolabeling. Anti-DNP mAb (E384C) (kappa/IgG1z), control conjugate 1, and DAR4 conjugate 4 were radiolabeled with

64Cu

and analyzed according to established

methods.60 NODA-GA functionalized Ab conjugates were incubated with

64CuCl 2

(1-2

mCi in 5-10 μL, 0.1 M HCl) at 37 °C for 1 hr in a thermomixer. The solution was challenged with 5 μL of 10 mM EDTA 37 °C for 5 min to remove nonspecifically bound 64Cu.

Labeling yield was determined by radiochemical thin layer chromatography (radio-

TLC). Briefly, 1-2 μL of reaction solution was applied to an ITLC-SG plate (Pall Corporation) and developed using a 1:1 mixture of 10% ammonium acetate and methanol. The TLC plate was then measured using a Bioscan AR-200 imaging scanner. 64Cu-labeled

conjugates were then passed through a Zeba spin desalting column (10K

MWOC) to remove EDTA and the small amount of unbound

64Cu

contained in the

sample. The radiochemical purity was found to be >95%, and labeled conjugates were combined with unlabeled conjugate to obtain the appropriate final mass dose (e.g., 30-40 mg/kg). In Vivo Imaging. PET/CT imaging was performed using a Siemens Inveon scanner. Animals were administered tail vein injections with 100 μCi of radiolabeled conjugate in 100-125 μL of saline.

Animals were anesthetized in a prone position using 1–2%

isoflurane delivered in 100% oxygen gas, provided with supplemental heating, and were 36 ACS Paragon Plus Environment


monitored for physiological signs throughout the duration of the 30 min PET scan acquisition.

Image reconstruction, attenuation correction, decay correction, co-

registration, and analysis were performed using Siemens IAW and IRW software applications.

Histamine-Induced Scratching in Mice. Subjects were C57Bl/6 male mice (Charles River Laboratories, Franklin, NY) aged between 8 and 10 weeks at the beginning of the study. One day prior to behavioral testing, mice were anesthetized under 3% isoflurane, and the area at the nape of the neck was shaved. Immediately afterward, mice were transported to the testing room and acclimated to individual sound-attenuated chambers (12" l × 9.5" w × 8.25" h, Med Associates VFC-008, NIR-022MD, St. Albans, VT) for 15 min. Twenty-four or seventy-two hours prior to histamine treatment, mice were subcutaneously administered conjugate 10 (25, 125, or 400 mg/kg body weight (n=8 per group)) or a vehicle control formulation (PBS with 1% MSA). Testing was performed the following day between the hours of 8 a.m. and 3 p.m. A separate group of animals was orally administered the antihistamine diphenhydramine (30 mg/kg in phosphate-buffered saline; Sigma D3630) 90 min prior to testing to serve as a positive control in each study. Histamine dichloride (8.15 mM in a volume of 100 μL; Sigma, H7250) was injected intradermally to the shaved area of all subjects, mice were placed into the soundattenuated testing chambers, and behavior was recorded on digital video files for a period of 15 min. Video recordings were later reviewed and individual scratching bouts were scored by trained experimenters. A scratching bout was defined as a rapid head tilt accompanied by a hind paw directed at the site of intradermal injection. Termination of a 37 ACS Paragon Plus Environment

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scratching bout was deemed to have occurred when the hind paw was placed back on the chamber floor or into the animal’s mouth. All dosing and scoring activities were conducted by experimenters who were fully blinded to treatment conditions.

Supporting Information The Supporting Information is available free of charge via the internet on the ACS Publications website at https://pubs.acs.org/. Full details of antibody sequence, conjugate characterization, biodistribution, calculated isoelectric points, pharmacokinetics, electrophysiology, additional peptide-antibody conjugates, pharmacodynamics, and open field locomotor activity in mice

Notes The authors declare the following competing financial interest(s): All authors are current or former employees of Amgen Inc.



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