Site-Specific Immuno-PET Tracer to Image PD-L1

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00010

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, ⁶⁴Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

Full text of DOI:10.1021/acs.molpharmaceut.9b00010

Subscriber access provided by La Trobe University Library
Article
A Site-Specific ImmunoPET Tracer to Image PD-L1
Haley L. Wissler, Emily B Ehlerding, Zhigang Lyu, Yue Zhao, Si Zhang, Anisa
Eshraghi, Zakey Yusuf Buuh, Jeffrey C McGuth, Yifu Guan, Jonathan W
Engle, Sarah J Bartlett, Vincent A. Voelz, Weibo Cai, and Rongsheng E. Wang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00010 • Publication Date (Web): 15 Mar 2019
Downloaded from http://pubs.acs.org on March 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
A Site-Specific ImmunoPET Tracer to Image PD-L1
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
Haley L. Wissler1,†, Emily B. Ehlerding2,†, Zhigang Lyu1, Yue Zhao1, Si Zhang1, Anisa Eshraghi1,
Zakey Yusuf Buuh1, Jeffrey C. McGuth1, Yifu Guan1, Jonathan W. Engle2, Sarah J. Bartlett1,
Vincent A. Voelz1, Weibo Cai2,*, Rongsheng E. Wang1,*
1. Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, PA, 19122, USA
2. Departments of Radiology and Medical Physics, University of Wisconsin-Madison, WI, 53705, USA
* To whom correspondence should be addressed:
Weibo Cai, Phone (608) 262-1749. Email: wcai@uwhealth.org
Rongsheng E. Wang, Phone (215) 204-1855. Email: rosswang@temple.edu
† These authors contributed equally.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 31
1
2
3
4
ABSTRACT
5
6
7
8
The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the
need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which
could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-
PET) allows for sensitive and quantitative imaging of target levels and has promising potential for
the noninvasive evaluation of immune checkpoint proteins. However, the advancement of
immuno-PET is currently limited by available imaging tools, which heavily rely on full-length
IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with
chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the
chelator NOTA, enabling radiolabeling for PET imaging, using the amber suppression-mediated
genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate
is homogeneous and demonstrated tight binding towards the PD-L1 antigen in vitro. The
radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for
effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models,
including the capturing of different PD-L1 expression levels in the spleens of the different mouse
types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific
tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-
specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which
could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in
immunotherapy.
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
KEYWORDS
Antibody, Immune checkpoint, Immunotherapy, PD-L1, Site-specific, Positron Emission
Tomography, Unnatural amino acid.
ACS Paragon Plus Environment

Page 3 of 31
Molecular Pharmaceutics
1
2
3
4
INTRODUCTION
5
6
7
8
Checkpoint inhibitor-based cancer immunotherapy has recently emerged as a pillar of effective
treatment to control tumor growth and dissemination.1, 2 The discovery of multiple immune
checkpoint mechanisms such as program death-ligand 1 (PD-L1), programmed cell death protein
1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) has facilitated the
understanding of how tumor cells bypass the attack of immune systems.3 Therapeutic treatments
utilizing immune checkpoint inhibitors would ‘wake up’ the previously suppressed immune cells
and activate them to attack tumor cells.3 Thus, these inhibitors such as antibodies, have become
first-line therapies for different tumor types.1, 3 Yet, heterogeneous outcomes in clinical responses,
frequent toxicity, and adverse effects necessitate the development of new methods for the
prognosis and monitoring of patient responses.1, 2, 4-8 A non-invasive strategy to comprehensively
monitor the expression of immune checkpoint proteins may help elucidate their roles in
immunotherapeutic responses, and potentially be useful in patient selection and treatment
monitoring.4, 9-13 Molecular imaging, especially positron emission tomography using antibodies
conjugated with radionuclides (Immuno-PET), allows for accurate and non-invasive evaluation of
target levels.14-16 Due to high sensitivity and resolution, immuno-PET has been successfully
applied to interrogate the relation between tissue uptake and clinical responses towards antibody
treatment.17-19 Thus, immuno-PET could be a crucial tool to test the biomarker hypothesis of
immune checkpoint proteins for immunotherapy.15, 16, 20
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
Despite previous PET imaging efforts for immune checkpoint proteins,9-11, 20-26 most immune-
checkpoint-based PET probes rely on full-length IgG antibodies,10, 21, 23, 24 which have limited
tissue/tumor penetrance. Fab fragments on the other hand, retain the binding of the antibody but
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 31
1
2
3
4
5
6
7
8
9
are devoid of the Fc effector domain. The resulting shorter circulation half-lives and less off-
target-related background signals make them ideal probe candidates.27, 28 Yet, few Fab-based PET
probes have been developed so far.27, 28 More importantly, common antibody probes have been
synthesized via random coupling of natural amino acids with chelators, resulting in heterogeneous
constructs with suboptimal stabilities, efficacies, and pharmacological properties.29, 30 Early
studies with cysteine-based site-specific immunoconjugates revealed higher target-to-background
imaging intensity ratios than random conjugates,30 demonstrating the positive effects on in vivo
performance of precise control of conjugation sites and stoichiometry. Yet, the suboptimal
biological stability of the resulting maleimdyl thioether bonds largely limited its broad
applications.30 Another cysteine-based approach, expressed protein ligation (EPL), could result in
relatively stable coupling, but the feasible site is mainly limited to C-terminals which may not
render the most stable conjugates.31 On the other hand, application of EPL to multiple
modifications within the protein sequence would be technically demanding.32, 33 Recently, another
site-specific conjugation strategy emerged, with the amber suppression-mediated genetic
incorporation of unnatural amino acids (UAA) during the translation of recombinant proteins.33, 34
In this approach, the amber stop codon employs paired orthogonal amino-acyl transferase and iso-
tRNA to incorporate the desired UAA site specifically into proteins including the recombinant
antibody.33, 34 Antibodies incorporating UAAs such as p-acetylphenylalanine (pAcF) can be
coupled to small molecules of interest via a stable linkage,29, 35, 36 generating site-specific
therapeutics in high yields. The UAA conjugates as therapeutic candidates have demonstrated
superior in vivo efficacy, stability, and toxicology profiles to the cysteine conjugates.34, 37, 38
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
Nevertheless, UAA-incorporated immunoconjugates have rarely been applied to PET imaging.29,
39
ACS Paragon Plus Environment

Page 5 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
To this end, we have developed a site-specific immuno-PET probe by incorporating one UAA, p-
azidophenylalanine (pAzF), into antibody Fab fragments. Using the antibody against the popularly
studied immune checkpoint protein PD-L1 as an example, we demonstrated for the first time the
generation, optimization, and in vivo PET imaging of a UAA-based site-specific Fab conjugate
with 1,4,7-triazacyclononane-N, N’,N”-triacetic acid (NOTA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Materials. Reagents and solvents were obtained directly from commercial resources such as
Thermo Fisher, VWR, and Sigma Aldrich, and were used without further purification. Analytical
thin-layer chromatography was performed with EMD Chemicals Silica gel 60 F254 plates, the
chemicals on which were visualized by UV lamp (Chemglass Life Sciences) at 254/365 nm,
KMnO4 staining, or PMA staining. For purification, flash column chromatography was performed
with silica gel grade 60 (230-400 mesh, Fisher Scientific). Preparative HPLC was carried out on
Waters 1525 series equipped with a 2489 UV/vis detector, 1525 binary pump, and a XBridge Prep
C18 column (5 µm, 19250 mm). Solvent A was water/0.5% trifluoroacetic acid (TFA), and
solvent B was acetonitrile. For mass spectrometry analysis, regular liquid chromatography-mass
spectrometry (LC-MS) was performed on Agilent 1100 series. High resolution LC-MS analysis
was done on an Agilent 6520 Accurate-Mass Quadrupole-Time-of-Flight (Q-TOF) coupled with
an electrospray ionization (ESI) source. A zorbax 300SB-C8 column (5µm, 4.650 mm) was used.
For acquisition of NMR spectroscopy, 1HNMR and 13CNMR were recorded on 400 MHz or 500
MHz Bruker Advance. The data were processed with the MestReNova Software, measuring signal
shifts in parts per million (ppm) downfield from the internal standard tetramethyl silane (TMS).
Methods. Please see the Supporting Information for experimental details.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 31
1
2
3
4
5
RESULTS AND DISCUSSION
6
7
8
9
Synthesis of the NOTA-BCN Linker. We began by designing and synthesizing the PET
chelator. Copper-64 as a metallic radionuclide garners interest due to its favorable physical
properties, appropriate half-life and ease of production.40-43 Among the available macrocyclic
chelates, the NOTA series have been reported to possess superior chelation ability.40 Notably, the
hexadentate NOTA derivative with 1-branched substitution (3p-C-NOTA) had higher
radiolabeling efficiency and stability than the other NOTA derivatives,40 but has yet to be utilized
for immuno-PET.44-46 Thus, we set out to synthesize a similar NOTA derivative with the termini
modified with bicyclo[6.1.0]non-4-yne (BCN) for strain-promoted azide-alkyne cycloaddition
(SPAAC) conjugation.47 6-benzamidohexanoic acid was first reacted with benzoyl chloride to
afford protected linker compound 1 (Scheme 1).48 Bromination followed by methylation resulted
in methyl 6-benzamido-2-bromohexanoate (compound 2). The 1,4,7-triazacyclononane (TACN)
moiety was introduced by reacting the linker compound 2 with TACN-1,7-bis(t-butyl acetate) in
the presence of potassium carbonate.45, 48 The resulting intermediate was hydrolyzed under strong
acid conditions to generate the amine derivatized NOTA compound 3. A final coupling of 3 with
the BCN-succinimidyl ester, followed by HPLC purification, afforded the NOTA-BCN linker
(compound 4) with > 95% purity, as shown by LC-MS analysis (Supplementary Information
Figure S1).
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
Design and Synthesis of Anti-PD-L1 Fab Mutant. Compared to IgGs, antibody Fab fragments
possess a small protein size thereby improving tissue penetrance, and the removal of the Fc portion
avoids unwanted effector effects.27, 28 Its shorter biological half-life enables labeling with shorter-
lived radionuclides such as copper-64, which taken together should lead to improved signal-to-
ACS Paragon Plus Environment

Page 7 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
background ratios at earlier time points.27 These unique characteristics make them not only
suitable for therapeutic development,49, 50 but also as ideal imaging agents,27 warranting a detailed
test of the site-specific incorporation of UAA. Among all the available immune checkpoint
proteins, PD-L1 appears to be the most studied due to its broad and dynamic expression profile
and its potential correlation with prognosis and therapy responsiveness.10, 20, 21, 23, 24, 26 Hence, we
picked the sequences coding for the variable regions of the αPD-L1 antibody avelumab, which has
been clinically approved for treating gastric cancer and Merkel-cell carcinoma, and is also human-
mouse cross-reactive.51 The plasmid (pBAD_αPD-L1) was constructed to harbor the heavy- and
lambda light-chain genes of the Fab fragment following a stll signal peptide.36, 52 The wild type
αPD-L1 Fab was expressed in E. coli, with a yield of 4.2 mg/L in shake flask cultures. SDS/PAGE
analysis showed that the Fab fragment migrated as a single band, with > 95% purity and a
molecular mass of ~ 45 kDa (Figure S2).
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
The site-specific labeling strategy based on the genetic incorporation of UAA allows one to
explore various conjugates with different geometries and stabilities.36, 52 Previous studies on a
couple of mutation sites have resulted in Fab fragments of significantly different yields.36
Nevertheless, there has been no systematic study done to determine the optimal conjugation site.
To this end, we selected 23 possible sites on αPD-L1 Fab, which are surface-exposed, distal to
binding sites, and are in flexible loops (Figure 1A). In silico screening was performed using the
RosettaBackrub algorithm to predict the viability of single point mutations at the selected sites
(Figure 2). The results suggested that mutations at HC-K129 would be most favorably tolerated
(Figure 2). Notably, the site HC-K129 has been experimentally explored before to be an ideal
conjugation site for pAcF with high expression yield, and little interference with antigen binding.36,
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 31
1
2
3
4
52, 53
As a negative control in our experimental validation of the screening results, LC-V202 was
5
6
7
8
picked for amber suppression as well. Despite the previous site-specific incorporation of pAcF
for oxime coupling,36 we decided to employ the incorporation of pAzF for strain-promoted
SPAAC in this study. Compared to oxime coupling, the strain-promoted SPAAC conjugation is
more bioorthogonal, and can proceed efficiently at neutral pH.39, 54, 55 Although there are few
reports in regards to its incorporation onto antibodies,54 electron-deficient aryl azides such as pAzF
have been shown to greatly accelerate the SPAAC reaction.47
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
Therefore, we performed site-directed mutation of HC-K129 or LC-V202 to the TAG codon,
and cotransformed each pBAD plasmid into the DH10B strain along with a plasmid
(pULTRA_pCNF)56 that encodes the polyspecific Mj-tRNA/tyrosyl-tRNA synthetase pair evolved
to incorporate the pAzF UAA. The expression yield for αPD-L1 Fab mutant (HC K129X, X =
pAzF) was 2.5 mg/L, while the yield for αPD-L1 Fab (LC V202X, X = pAzF) was only 0.04 mg/L.
This significant difference is consistent with the in-silico screening results, and emphasizes the
importance of optimizing the labeling site. In parallel, we also cloned and expressed αHER2 Fab
mutant (HC K129X, X = pAzF), which could serve as a non-relevant antibody control for follow-
up imaging experiments. Both Fab antibodies migrated as a single band on SDS/PAGE analysis,
indicating a > 95% purity, and a molecular weight of ~ 45 kDa (Figure S2). Their identities were
further confirmed by ESI-MS analysis (Figure S3).
Synthesis and Characterization of Site-Specific NOTA-Anti-PD-L1 Fab Conjugate. Next,
we conjugated the purified Fab mutants (αPD-L1 Fab and αHER2 Fab, HC K129X, X = pAzF)
with linker compound 4 (Figure 1A). Each pAzF-containing Fab was reacted with a 10-fold
ACS Paragon Plus Environment

Page 9 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
o
excess of NOTA-BCN linker at 37 C for 12 h to ensure the complete conjugation. The Fab
conjugates were purified following the reported procedures,36, 57 and analyzed by SDS/PAGE
(Figure S2) and ESI-MS (Figure S3), which demonstrated that the final conjugates were > 95%
pure, and had the desired molecular mass with a NOTA-to-antibody ratio of 1 (Figure S3). The
binding affinity of αPD-L1 Fab fragments to PD-L1 was evaluated by ELISA (Figure 1B). The
conjugate and the HC K129 mutant both have a similar affinity (EC50’s ~ 0.7 nM) to the wild type,
suggesting that neither the site-directed mutation nor payload conjugation at the selected site
compromises the antibody’s binding.
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
In vivo PET Imaging of PD-L1 Expression. To utilize this class of site-specific Fab conjugates
as imaging probes, we evaluated their in vivo PET imaging capability using nude mice that are
immuno-deficient and can potentially carry various tumor types for future studies.58-60 After
radiolabeling with 64Cu, 50 – 70 µCi of either 64Cu-NOTA-αPD-L1 or 64Cu-NOTA-αHER2 were
intravenously administered to nude mice. PET scans were performed at 5, 15, and 45 min
postinjection (p.i.). As demonstrated by Figure 3A, PET signal vs. noise became stable at 45 min
p.i., with background signals almost cleared. Compared to the intact full length antibody
counterparts,10, 23 Fab fragment-based PET agents provided higher contrast imaging due to the low
level of nonspecific accumulation and faster clearance mechanisms,61 potentially ideal for imaging
highly abundant surface markers. Fab conjugates were mostly cleared through the renal pathway
and excreted via the urinary bladder, as evidenced by the extremely high kidney and bladder
uptakes. Both images (Figure 3A) and region-of-interest (ROI) analysis (Figure 3B, 3C) revealed
specific uptake of 64Cu-NOTA-αPD-L1 in brown adipose tissue (BAT) and spleen. For BAT, the
accumulation of 64Cu-NOTA-αPD-L1 was highest immediately after injection (6.2 ± 2.4 %ID/g,
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 31
1
2
3
4
5
6
7
8
9
64
n = 3) and remained high throughout the study (6.1 ± 2.1 %ID/g at 45 min). The control Cu-
NOTA-αHER2, on the contrary, displayed minimal enrichment in BAT, from 1.7 ± 0.2 %ID/g at
15 min to 1.6 ± 0.1 %ID/g at 45 min (n = 3). For the spleen, there was a similar trend for 64Cu-
NOTA-αPD-L1 (11.8 ± 3.0 %ID/g at 15 min to 12.0 ± 1.9 %ID/g at 45 min, n = 3), while the
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
64
uptake of the control Cu-NOTA-αHER2 (2.5 ± 0.1 %ID/g at 15 min to 2.1 ± 0.3 %ID/g at 45
min, n = 3) remained low. These resulted in statistically significant differences (p < 0.05) in both
BAT and spleen uptake for the two imaging tracers at the time point of 45 min p.i. (Figure 3B,
3C), while there was no notable difference in other analyzed organs.
To further confirm that the observed enrichment of 64Cu-NOTA-αPD-L1 is antigen-specific, we
performed blocking studies in nude mice by injecting wild type αPD-L1 Fab at a dose of ~ 200
µg/mouse prior to the administration of the corresponding NOTA conjugate. As shown in Figures
3B and 3C, the uptake of the NOTA conjugate was drastically decreased in both BAT (1.9 ±
0.3 %ID/g at 15 min and 1.4 ± 0.3 %ID/g at 45 min, n = 3) and spleen (3.8 ± 0.3 %ID/g at 15 min
and 1.7 ± 1.2 %ID/g at 45 min, n = 3), similar to the levels of the control αHER2 conjugate. Taken
together, this set of results indicate that the uptake of PET tracer in BAT and spleen of nude mice
was PD-L1 specific. This finding suggests that the 64Cu-NOTA-αPD-L1 probe is a reliable, non-
metabolic tracer for in vivo PD-L1 expression. PD-L1 checkpoint was initially regarded to be
primarily expressed in the extralymphatic organs.3, 10 The existence of PD-L1 in the spleen is
consistent with recent findings that PD-L1 is additionally expressed in secondary lymphatic
organs.10 The presence of PD-L1 in BAT further confirms that adipose tissue is influenced by
arms of the immune system.10, 21, 62 These organs’ expression of PD-L1 may suggest their
utilization of PD-1/PD-L1 interactions to locally suppress unwanted T-cell responses.10
ACS Paragon Plus Environment

Page 11 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
Next, we set out to gauge the general applicability of our imaging probe in another mouse model,
C57BL/6 mice that are immunocompetent. Similarly, the BAT and spleen were found to have
9
64
64
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
much higher accumulation of Cu-NOTA-αPD-L1 than that of the control Cu-NOTA-αHER2
throughout the study (Figure S4). The uptake of 64Cu-NOTA-αPD-L1 in the spleen was less
significant (Figures S4A, S4C), presumably due to the more varied expression levels of PD-L1 in
the spleens among individual C57BL/6 mice (Figure S4A, S4C).10, 63, 64 This observation may
accurately reflect the real situation, which in translation to the clinical situation, may partially
account for heterogeneous immunotherapeutic responses.
Ex vivo Biodistribution Studies and Immunofluorescence Staining. Following the last PET
scan at 90 min p.i., C57BL/6 mice were sacrificed, with major organs resected for ex vivo gamma
counting to corroborate the quantification of PET images (Figure S5). The kidneys from both
groups had extremely high accumulation of the tracers, at 157 ± 27 %ID/g for 64Cu-NOTA-αPD-
64
L1 and 219 ± 28 %ID/g for Cu-NOTA-αHer2. Low but specific tracer uptake in the lung and
intestines were also revealed, consistent with the reported PD-L1 expression in minor cell
populations.10 Notably, the biodistribution data corroborated the findings from in vivo PET ROI
analysis. The BAT uptakes with the two imaging agents were significantly different from one
another in this analysis as well (4.5 ± 1.5 %ID/g for 64Cu-NOTA-αPD-L1 and 0.9 ± 0.2 %ID/g for
64Cu-NOTA-αHER2, p<0.05). The enrichment of 64Cu-NOTA-αPD-L1 was still higher than the
control in spleen (9.4 ± 3.9 %ID/g vs. 2.3 ± 0.8 %ID/g), but with large variations. Follow-up
immunofluorescent staining of these organs further confirmed that both BAT and spleen are PD-
L1 positive (Figure 4). These results are consistent with recent literature reports,9, 10, 21, 24, 26 which
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 31
1
2
3
4
suggests the tissue-specific (BAT, spleen) uptake of antibodies against PD-L1, and indicates that
5
6
our 64Cu-NOTA-αPD-L1 probe is highly specific towards PD-L1.
7
8
9
64
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
In Conclusion: We have developed and studied a Cu-labeled, UAA-based, site-specific Fab
conjugate as an imaging probe to measure PD-L1 expression levels in vivo with immunoPET. This
antibody conjugate was optimized at a fixed site and stoichiometry, and bears an indistinguishable
binding affinity from the unconjugated wild type towards the cognate antigen. When applied to
noninvasive in vivo imaging, the probe can sensitively detect the expression levels of the targeted
antigen, in different mouse models. The particular PD-L1 expression on non-tumor organs, such
as BAT, lung, and intestines, as revealed by this probe, may indicate that targeted T cell responses
in these organs are strongly suppressed by the PD1/PD-L1 immune checkpoint.10 Further, these
findings may explain the frequent association of immune checkpoint blockade with immune-
related adverse effects on these organs,10 underlying the importance of image-guided prognosis
and treatment monitoring in immunotherapy. These data generally support the hypothesis that
imaging PD-L1 expression with UAA-based site-specific Fab conjugates may be feasible in future
clinical settings. Further evaluation of the conjugate in disease-related models (xenograft and
syngeneic tumor models) will be required to determine its clinical potential. In addition, we are
comparing the properties and activity of this conjugate with random conjugates and cysteine-based
site-specific conjugates. Finally, this work suggests that the amber-suppression mediated genetic
incorporation strategy has applicability as a route to a class of site-specific immuno-PET probes
that can potentially guide immune checkpoint-targeted immunotherapy.
ASSOCIATED CONTENT
ACS Paragon Plus Environment

Page 13 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Supporting Figures (S1 – S5) and detailed descriptions of the experimental methods applied in this
study (PDF).
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
AUTHOR INFORMATION
Corresponding Authors
*Phone: 608-262-1749, E-mail: wcai@uwhealth.org
*Phone: 215-204-1855, E-mail: rosswang@temple.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by grant #15-175-22 from the American Cancer Society, Temple
University Startup Fund, and was also supported, in part, by the University of Wisconsin-Madison
and the National Institutes of Health (P30CA014520, T32GM008505, T32CA009206). V.V. and
S.Z. were supported by National Institutes of Health (NIH) grant 1R01GM123296.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 31
1
2
3
4
REFERENCES
5
6
7
(1) Burugu, S., Dancsok, A. R., and Nielsen, T. O. (2018) Emerging targets in cancer
immunotherapy, Semin. Cancer Biol. 52(Pt 2): 39-52.
8
9
(2) Davies, M., and Duffield, E. A. (2017) Safety of checkpoint inhibitors for cancer treatment:
strategies for patient monitoring and management of immune-mediated adverse events,
Immunotargets Ther. 6, 51-71.
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
(3) Pardoll, D. M. (2012) The blockade of immune checkpoints in cancer immunotherapy, Nat Rev
Cancer 12, 252-264.
(4) Aris, M., Mordoh, J., and Barrio, M. M. (2017) Immunomodulatory Monoclonal Antibodies in
Combined Immunotherapy Trials for Cutaneous Melanoma, Front. Immunol. 8, 1024.
(5) Voutsadakis, I. A. (2016) Immune Blockade Inhibition in Breast Cancer, Anticancer Res. 36,
5607-5622.
(6) Moslehi, J. J., Salem, J. E., Sosman, J. A., Lebrun-Vignes, B., and Johnson, D. B. (2018)
Increased reporting of fatal immune checkpoint inhibitor-associated myocarditis, Lancet. 391, 933.
(7) Johnson, D. B., Balko, J. M., Compton, M. L., Chalkias, S., Gorham, J., Xu, Y., Hicks, M.,
Puzanov, I., Alexander, M. R., Bloomer, T. L., Becker, J. R., Slosky, D. A., Phillips, E. J.,
Pilkinton, M. A., Craig-Owens, L., Kola, N., Plautz, G., Reshef, D. S., Deutsch, J. S., Deering, R.
P., Olenchock, B. A., Lichtman, A. H., Roden, D. M., Seidman, C. E., Koralnik, I. J., Seidman, J.
G., Hoffman, R. D., Taube, J. M., Diaz, L. A., Jr., Anders, R. A., Sosman, J. A., and Moslehi, J. J.
(2016) Fulminant Myocarditis with Combination Immune Checkpoint Blockade, N. Engl. J. Med.
375, 1749-1755.
(8) Leem, G., and Song, S. Y. (2016) Immunotherapy in Pancreatic Cancer; the Road Less
Traveled, Immunol. Disord. Immunother. 1, 1000106.
(9) Ingram, J. R., Dougan, M., Rashidian, M., Knoll, M., Keliher, E. J., Garrett, S., Garforth, S.,
Blomberg, O. S., Espinosa, C., Bhan, A., Almo, S. C., Weissleder, R., Lodish, H., Dougan, S. K.,
and Ploegh, H. L. (2017) PD-L1 is an activation-independent marker of brown adipocytes, Nat.
Commun. 8, 647.
(10) Hettich, M., Braun, F., Bartholoma, M. D., Schirmbeck, R., and Niedermann, G. (2016) High-
Resolution PET Imaging with Therapeutic Antibody-based PD-1/PD-L1 Checkpoint Tracers,
Theranostics 6, 1629-1640.
(11) Natarajan, A., Mayer, A. T., Xu, L., Reeves, R. E., Gano, J., and Gambhir, S. S. (2015) Novel
Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating
Lymphocytes, Bioconjug. Chem. 26, 2062-2069.
ACS Paragon Plus Environment

Page 15 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
(12) Rom-Jurek, E. M., Kirchhammer, N., Ugocsai, P., Ortmann, O., Wege, A. K., and Brockhoff,
G. (2018) Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell
Lines In Vitro and in Immunodeficient and Humanized Tumor Mice, Int. J. Mol. Sci. 19, pii:E563.
7
8
9
(13) Sabatier, R., Finetti, P., Mamessier, E., Adelaide, J., Chaffanet, M., Ali, H. R., Viens, P.,
Caldas, C., Birnbaum, D., and Bertucci, F. (2015) Prognostic and predictive value of PDL1
expression in breast cancer, Oncotarget 6, 5449-5464.
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
(14) Ehlerding, E. B., England, C. G., McNeel, D. G., and Cai, W. (2016) Molecular Imaging of
Immunotherapy Targets in Cancer, J. Nucl. Med. 57, 1487-1492.
(15) Bauckneht, M., Piva, R., Sambuceti, G., Grossi, F., and Morbelli, S. (2017) Evaluation of
response to immune checkpoint inhibitors: Is there a role for positron emission tomography?,
World J. Radiol. 9, 27-33.
(16) Widmann, G., Nguyen, V. A., Plaickner, J., and Jaschke, W. (2016) Imaging Features of
Toxicities by Immune Checkpoint Inhibitors in Cancer Therapy, Curr. Radiol. Rep. 5, 59.
(17) Huisman, M., Menke, C., Gootjes, E., Vugts, D., Greuter, H., Boellaard, R., Verheul, H., and
Van Dongen, G. (2014) 89Zr labeled cetuximab imaging in colorectal cancer patients aimed at
image-guided patient selection for anti-EGFR treatment, J. Nucl. Med. 55, 225.
(18) Menke-van der Houven van Oordt, C. W., Gootjes, E. C., Huisman, M. C., Vugts, D. J., Roth,
C., Luik, A. M., Mulder, E. R., Schuit, R. C., Boellaard, R., Hoekstra, O. S., van Dongen, G. A.,
and Verheul, H. M. (2015) 89Zr-cetuximab PET imaging in patients with advanced colorectal
cancer, Oncotarget 6, 30384-30393.
(19) van Dijk, L. K., Boerman, O. C., Kaanders, J. H., and Bussink, J. (2015) PET Imaging in
Head and Neck Cancer Patients to Monitor Treatment Response: A Future Role for EGFR-
Targeted Imaging, Clin. Cancer Res. 21, 3602-3609.
(20) Chatterjee, S., Lesniak, W. G., and Nimmagadda, S. (2017) Noninvasive Imaging of Immune
Checkpoint Ligand PD-L1 in Tumors and Metastases for Guiding Immunotherapy, Mol. Imaging
16, 1536012117718459.
(21) Chatterjee, S., Lesniak, W. G., Gabrielson, M., Lisok, A., Wharram, B., Sysa-Shah, P., Azad,
B. B., Pomper, M. G., and Nimmagadda, S. (2016) A humanized antibody for imaging immune
checkpoint ligand PD-L1 expression in tumors, Oncotarget 7, 10215-10227.
(22) England, C. G., Jiang, D., Ehlerding, E. B., Rekoske, B. T., Ellison, P. A., Hernandez, R.,
Barnhart, T. E., McNeel, D. G., Huang, P., and Cai, W. (2018) (89)Zr-labeled nivolumab for
imaging of T-cell infiltration in a humanized murine model of lung cancer, Eur. J. Nucl. Med. Mol.
Imaging 45, 110-120.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 31
1
2
3
4
5
6
(23) Heskamp, S., Hobo, W., Molkenboer-Kuenen, J. D., Olive, D., Oyen, W. J., Dolstra, H., and
Boerman, O. C. (2015) Noninvasive Imaging of Tumor PD-L1 Expression Using Radiolabeled
Anti-PD-L1 Antibodies, Cancer Res. 75, 2928-2936.
7
8
9
(24) Josefsson, A., Nedrow, J. R., Park, S., Banerjee, S. R., Rittenbach, A., Jammes, F., Tsui, B.,
and Sgouros, G. (2016) Imaging, Biodistribution, and Dosimetry of Radionuclide-Labeled PD-L1
Antibody in an Immunocompetent Mouse Model of Breast Cancer, Cancer Res. 76, 472-479.
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
(25) Natarajan, A., Mayer, A. T., Reeves, R. E., Nagamine, C. M., and Gambhir, S. S. (2017)
Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on
Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model, Mol. Imaging Biol. 19, 903-914.
(26) Truillet, C., Oh, H. L. J., Yeo, S. P., Lee, C. Y., Huynh, L. T., Wei, J., Parker, M. F. L.,
Blakely, C., Sevillano, N., Wang, Y. H., Shen, Y. S., Olivas, V., Jami, K. M., Moroz, A., Jego, B.,
Jaumain, E., Fong, L., Craik, C. S., Chang, A. J., Bivona, T. G., Wang, C. I., and Evans, M. J.
(2018) Imaging PD-L1 Expression with ImmunoPET, Bioconjug. Chem. 29, 96-103.
(27) Ilovich, O., Natarajan, A., Hori, S., Sathirachinda, A., Kimura, R., Srinivasan, A., Gebauer,
M., Kruip, J., Focken, I., Lange, C., Carrez, C., Sassoon, I., Blanc, V., Sarkar, S. K., and Gambhir,
S. S. (2015) Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic
Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope, Radiology 276, 191-198.
(28) Maute, R. L., Gordon, S. R., Mayer, A. T., McCracken, M. N., Natarajan, A., Ring, N. G.,
Kimura, R., Tsai, J. M., Manglik, A., Kruse, A. C., Gambhir, S. S., Weissman, I. L., and Ring, A.
M. (2015) Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-
PET imaging, Proc. Natl. Acad. Sci. U S A 112, E6506-6514.
(29) Adumeau, P., Sharma, S. K., Brent, C., and Zeglis, B. M. (2016) Site-Specifically Labeled
Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids, Mol.
Imaging Biol. 18, 153-165.
(30) Adumeau, P., Sharma, S. K., Brent, C., and Zeglis, B. M. (2016) Site-Specifically Labeled
Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans, Mol. Imaging
Biol. 18, 1-17.
(31) Frutos, S., Hernandez, J. L., Otero, A., Calvis, C., Adan, J., Mitjans, F., and Vila-Perello, M.
(2018) Site-Specific Antibody Drug Conjugates Using Streamlined Expressed Protein Ligation,
Bioconjug. Chem. 29, 3503-3508.
(32) Muir, T. W. (2003) Semisynthesis of proteins by expressed protein ligation, Annu. Rev.
Biochem. 72, 249-289.
(33) Wang, L., Xie, J., and Schultz, P. G. (2006) Expanding the genetic code, Annu. Rev. Biophys.
Biomol. Struct. 35, 225-249.
ACS Paragon Plus Environment

Page 17 of 31
Molecular Pharmaceutics
1
2
3
4
5
(34) Liu, C. C., and Schultz, P. G. (2010) Adding new chemistries to the genetic code, Annu. Rev.
Biochem. 79, 413-444.
6
7
8
9
(35) Cao, Y., Axup, J. Y., Ma, J. S., Wang, R. E., Choi, S., Tardif, V., Lim, R. K., Pugh, H. M.,
Lawson, B. R., Welzel, G., Kazane, S. A., Sun, Y., Tian, F., Srinagesh, S., Javahishvili, T., Schultz,
P. G., and Kim, C. H. (2015) Multiformat T-cell-engaging bispecific antibodies targeting human
breast cancers, Angew. Chem. Int. Ed. Engl. 54, 7022-7027.
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
(36) Lyu, Z., Kang, L., Buuh, Z. Y., Jiang, D., McGuth, J. C., Du, J., Wissler, H. L., Cai, W., and
Wang, R. E. (2018) A Switchable Site-Specific Antibody Conjugate, ACS Chem. Biol. 13, 958-
964.
(37) Jackson, D., Atkinson, J., Guevara, C. I., Zhang, C., Kery, V., Moon, S. J., Virata, C., Yang,
P., Lowe, C., Pinkstaff, J., Cho, H., Knudsen, N., Manibusan, A., Tian, F., Sun, Y., Lu, Y., Sellers,
A., Jia, X. C., Joseph, I., Anand, B., Morrison, K., Pereira, D. S., and Stover, D. (2014) In vitro
and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates,
PLoS One 9, e83865.
(38) Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., Phuong, T., Barnett, R., Hehli,
B., Song, F., DeGuzman, M. J., Ensari, S., Pinkstaff, J. K., Sullivan, L. M., Biroc, S. L., Cho, H.,
Schultz, P. G., DiJoseph, J., Dougher, M., Ma, D., Dushin, R., Leal, M., Tchistiakova, L., Feyfant,
E., Gerber, H. P., and Sapra, P. (2014) A general approach to site-specific antibody drug
conjugates, Proc. Natl. Acad. Sci. U S A 111, 1766-1771.
(39) Wu, Y., Zhu, H., Zhang, B., Liu, F., Chen, J., Wang, Y., Wang, Y., Zhang, Z., Wu, L., Si, L.,
Xu, H., Yao, T., Xiao, S., Xia, Q., Zhang, L., Yang, Z., and Zhou, D. (2016) Synthesis of Site-
Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion,
Bioconjug. Chem. 27, 2460-2468.
(40) Wu, N., Kang, C. S., Sin, I., Ren, S., Liu, D., Ruthengael, V. C., Lewis, M. R., and Chong,
H. S. (2016) Promising bifunctional chelators for copper 64-PET imaging: practical (64)Cu
radiolabeling and high in vitro and in vivo complex stability, J. Biol. Inorg. Chem. 21, 177-184.
(41) Shokeen, M., and Anderson, C. J. (2009) Molecular imaging of cancer with copper-64
radiopharmaceuticals and positron emission tomography (PET), Acc. Chem. Res. 42, 832-841.
(42) Nayak, T. K., and Brechbiel, M. W. (2009) Radioimmunoimaging with longer-lived positron-
emitting radionuclides: potentials and challenges, Bioconjug. Chem. 20, 825-841.
(43) Cutler, C. S., Hennkens, H. M., Sisay, N., Huclier-Markai, S., and Jurisson, S. S. (2013)
Radiometals for combined imaging and therapy, Chem. Rev. 113, 858-883.
(44) de Sa, A., Bonnet, C. S., Geraldes, C. F., Toth, E., Ferreira, P. M., and Andre, J. P. (2013)
Thermodynamic stability and relaxation studies of small, triaza-macrocyclic Mn(II) chelates,
Dalton Trans. 42, 4522-4532.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 31
1
2
3
4
5
6
(45) Mate, G., Simecek, J., Pniok, M., Kertesz, I., Notni, J., Wester, H. J., Galuska, L., and
Hermann, P. (2015) The influence of the combination of carboxylate and phosphinate pendant
arms in 1,4,7-triazacyclononane-based chelators on their 68Ga labelling properties, Molecules 20,
13112-13126.
7
8
9
(46) Riss, P. J., Kroll, C., Nagel, V., and Rosch, F. (2008) NODAPA-OH and NODAPA-(NCS)n:
synthesis, 68Ga-radiolabelling and in vitro characterisation of novel versatile bifunctional
chelators for molecular imaging, Bioorg. Med. Chem. Lett. 18, 5364-5367.
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
(47) Dommerholt, J., van Rooijen, O., Borrmann, A., Guerra, C. F., Bickelhaupt, F. M., and van
Delft, F. L. (2014) Highly accelerated inverse electron-demand cycloaddition of electron-deficient
azides with aliphatic cyclooctynes, Nat. Commun. 5, 5378.
(48) Stasiuk, G. J., Tamang, S., Imbert, D., Poillot, C., Giardiello, M., Tisseyre, C., Barbier, E. L.,
Fries, P. H., de Waard, M., Reiss, P., and Mazzanti, M. (2011) Cell-permeable Ln(III) chelate-
functionalized InP quantum dots as multimodal imaging agents, ACS Nano 5, 8193-8201.
(49) Badescu, G., Bryant, P., Bird, M., Henseleit, K., Swierkosz, J., Parekh, V., Tommasi, R.,
Pawlisz, E., Jurlewicz, K., Farys, M., Camper, N., Sheng, X., Fisher, M., Grygorash, R., Kyle, A.,
Abhilash, A., Frigerio, M., Edwards, J., and Godwin, A. (2014) Bridging disulfides for stable and
defined antibody drug conjugates, Bioconjug. Chem. 25, 1124-1136.
(50) Qi, J., Ye, X., Ren, G., Kan, F., Zhang, Y., Guo, M., Zhang, Z., and Li, D. (2014)
Pharmacological efficacy of anti-IL-1beta scFv, Fab and full-length antibodies in treatment of
rheumatoid arthritis, Mol. Immunol. 57, 59-65.
(51) Boyerinas, B., Jochems, C., Fantini, M., Heery, C. R., Gulley, J. L., Tsang, K. Y., and Schlom,
J. (2015) Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody
Avelumab (MSB0010718C) on Human Tumor Cells, Cancer Immunol. Res. 3, 1148-1157.
(52) Kim, C. H., Axup, J. Y., Lawson, B. R., Yun, H., Tardif, V., Choi, S. H., Zhou, Q., Dubrovska,
A., Biroc, S. L., Marsden, R., Pinstaff, J., Smider, V. V., and Schultz, P. G. (2013) Bispecific small
molecule-antibody conjugate targeting prostate cancer, Proc. Natl. Acad. Sci. U S A 110, 17796-
17801.
(53) Axup, J. Y., Bajjuri, K. M., Ritland, M., Hutchins, B. M., Kim, C. H., Kazane, S. A., Halder,
R., Forsyth, J. S., Santidrian, A. F., Stafin, K., Lu, Y., Tran, H., Seller, A. J., Biroc, S. L., Szydlik,
A., Pinkstaff, J. K., Tian, F., Sinha, S. C., Felding-Habermann, B., Smider, V. V., and Schultz, P.
G. (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids, Proc.
Natl. Acad. Sci. U S A 109, 16101-16106.
(54) Kern, J. C., Cancilla, M., Dooney, D., Kwasnjuk, K., Zhang, R., Beaumont, M., Figueroa, I.,
Hsieh, S., Liang, L., Tomazela, D., Zhang, J., Brandish, P. E., Palmieri, A., Stivers, P., Cheng, M.,
Feng, G., Geda, P., Shah, S., Beck, A., Bresson, D., Firdos, J., Gately, D., Knudsen, N., Manibusan,
A., Schultz, P. G., Sun, Y., and Garbaccio, R. M. (2016) Discovery of Pyrophosphate Diesters as
ACS Paragon Plus Environment

Page 19 of 31
Molecular Pharmaceutics
1
2
3
4
5
Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody-Drug Conjugates, J. Am.
Chem. Soc. 138, 1430-1445.
6
7
(55) Oller-Salvia, B. (2018) Genetic Encoding of a Non-Canonical Amino Acid for the Generation
of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction, J. Vis. Exp. (139).
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
(56) Young, D. D., Young, T. S., Jahnz, M., Ahmad, I., Spraggon, G., and Schultz, P. G. (2011)
An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity, Biochemistry 50,
1894-1900.
(57) Wang, R. E., Liu, T., Wang, Y., Cao, Y., Du, J., Luo, X., Deshmukh, V., Kim, C. H., Lawson,
B. R., Tremblay, M. S., Young, T. S., Kazane, S. A., Wang, F., and Schultz, P. G. (2015) An
immunosuppressive antibody-drug conjugate, J. Am. Chem. Soc. 137, 3229-3232.
(58) Ehlerding, E. B., England, C. G., Jiang, D., Graves, S. A., Kang, L., Lacognata, S., Barnhart,
T. E., and Cai, W. (2017) CD38 as a PET Imaging Target in Lung Cancer, Mol. Pharm. 14, 2400-
2406.
(59) Fliedner, F. P., Hansen, A. E., Jorgensen, J. T., and Kjaer, A. (2016) The use of matrigel has
no influence on tumor development or PET imaging in FaDu human head and neck cancer
xenografts, BMC Med. Imaging 16, 5.
(60) Luo, H., England, C. G., Goel, S., Graves, S. A., Ai, F., Liu, B., Theuer, C. P., Wong, H. C.,
Nickles, R. J., and Cai, W. (2017) ImmunoPET and Near-Infrared Fluorescence Imaging of
Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment, Mol. Pharm. 14, 1646-
1655.
(61) Olafsen, T., and Wu, A. M. (2010) Antibody vectors for imaging, Semin. Nucl. Med. 40, 167-
181.
(62) DiSpirito, J. R., and Mathis, D. (2015) Immunological contributions to adipose tissue
homeostasis, Semin. Immunol. 27, 315-321.
(63) Nedrow, J. R., Josefsson, A., Park, S., Ranka, S., Roy, S., and Sgouros, G. (2017) Imaging of
Programmed Cell Death Ligand 1: Impact of Protein Concentration on Distribution of Anti-PD-
L1 SPECT Agents in an Immunocompetent Murine Model of Melanoma, J. Nucl. Med. 58, 1560-
1566.
(64) Tang, H., Liang, Y., Anders, R. A., Taube, J. M., Qiu, X., Mulgaonkar, A., Liu, X.,
Harrington, S. M., Guo, J., Xin, Y., Xiong, Y., Nham, K., Silvers, W., Hao, G., Sun, X., Chen, M.,
Hannan, R., Qiao, J., Dong, H., Peng, H., and Fu, Y. X. (2018) PD-L1 on host cells is essential for
PD-L1 blockade-mediated tumor regression, J. Clin. Invest. 128, 580-588.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 31
1
2
3
4
5
6
Figure Legends:
7
8
9
Scheme 1. Synthetic scheme of 1,4,7-triazacyclononane-N, N’, N”–triacetic acid (NOTA)
derivative with a (1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-ylmethylcarbamate (BCN) linker.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Synthesis and characterization of the site-specific 64Cu-NOTA-anti-PD-L1 Fab
conjugate. (A) In silico screening with Rosetta has been performed with 23 solvent-exposed sites
to determine the optimal mutation site, followed by site-directed mutagenesis to generate the anti-
PD-L1 Fab (HC K129X, X = pAzF). Conjugation of this mutant with linker compound 4 will
afford site-specific NOTA-anti-PD-L1 Fab conjugate 5 and its radiolabeled version, 6. (B) ELISA
analysis of the binding affinities of anti-PD-L1 Fab fragments. The EC50 was calculated to be 7.8
x 10-10 M for wild type, 7.3 x 10-10 M for the mutant (HC K129X, X = pAzF), and 7.2 x 10-10
M
for conjugate 5.
Figure 2. In silico screening of mutation sites on αPD-L1 Fab. (A) Changes in stability ∆REU
(Rosetta energy units) for single-residue mutations to alanine (red) and phenylalanine (blue). Error
bars represent standard deviations in ∆REU across the twenty structures given by the
RosettaBackrub algorithm. (B) Similar changes in stability were predicted for alanine and
phenylalanine. (C) Cumulative histograms of the predicted score show that mutations at (heavy
chain) H_K129 are predicted to be the most favorable of the 23 selected residues. Predictions for
the chosen negative control (light chain) L_V202, in contrast, are not ranked as especially
favorable.
64
64
Figure 3. In vivo PET imaging studies with Cu-NOTA-αHER2 and Cu-NOTA-αPD-L1 in
nude mice. (A) PET scans at 15, and 45 min p.i. of 64Cu-NOTA-αHER2 (left), 64Cu-NOTA-αPD-
L1 (middle), or 64Cu-NOTA-αPD-L1 with preblocking by αPD-L1 w.t. (right); yellow arrowhead
indicates brown fat while white arrowhead points to the spleen. (B) Tracer uptake (%ID/g) for the
three imaging groups in brown fat based on quantitative region-of-interest (ROI) analysis of the
PET images. (C) Tracer uptake (%ID/g) in spleen. “*” represents P < 0.05. n = 3.
Figure 4. Immunofluorescent staining of brown adipose tissue for PD-L1 expression (red), and
spleen tissue for PD-L1 (cyan) and CD45 (green) expressions. Nuclei (blue) were stained as
controls.
ACS Paragon Plus Environment

Page 21 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
O
O
Br
BzCl,
1)
2)
OH
P, Br2
OMe
OH
N
H
N
H2N
H
aq. NaOH
CH3OH
O
O
O
9
1
2
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
O
H
HO
N
N
O
O
/ K2CO3, ACN
O
N
N
1)
2)
tBuO
OtBu
N
N
NH2
HO
6 M HCl
HO
O
3
O
HO
H
O
N
O
O
H
N
O
H
H
O
N
N
N
O
HO
O
H
O
N-methylmorpholine, DMF
HO
O
4
Scheme 1 – Synthetic scheme of 1,4,7-triazacyclononane-N, N’, N”–triacetic acid (NOTA)
derivative with a (1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-ylmethylcarbamate (BCN) linker.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 31
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Synthesis and characterization of the site-specific 64Cu-NOTA-anti-PD-L1 Fab
conjugate. (A) In silico screening with Rosetta has been performed with 23 solvent-exposed sites
to determine the optimal mutation site, followed by site-directed mutagenesis to generate the anti-
PD-L1 Fab (HC K129X, X = pAzF). Conjugation of this mutant with linker compound 4 will
afford site-specific NOTA-anti-PD-L1 Fab conjugate 5 and its radiolabeled version, 6. (B) ELISA
analysis of the binding affinities of anti-PD-L1 Fab fragments. The EC50 was calculated to be 7.8
x 10-10 M for wild type, 7.3 x 10-10 M for the mutant (HC K129X, X = pAzF), and 7.2 x 10-10
M
for conjugate 5.
ACS Paragon Plus Environment

Page 23 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. In silico screening of mutation sites on αPD-L1 Fab. (A) Changes in stability ∆REU
(Rosetta energy units) for single-residue mutations to alanine (red) and phenylalanine (blue). Error
bars represent standard deviations in ∆REU across the twenty structures given by the
RosettaBackrub algorithm. (B) Similar changes in stability were predicted for alanine and
phenylalanine. (C) Cumulative histograms of the predicted score show that mutations at (heavy
chain) H_K129 are predicted to be the most favorable of the 23 selected residues. Predictions for
the chosen negative control (light chain) L_V202, in contrast, are not ranked as especially
favorable.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 31
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
64
64
Figure 3. In vivo PET imaging studies with Cu-NOTA-αHER2 and Cu-NOTA-αPD-L1 in
nude mice. (A) PET scans at 15, and 45 min p.i. of 64Cu-NOTA-αHER2 (left), 64Cu-NOTA-αPD-
L1 (middle), or 64Cu-NOTA-αPD-L1 with preblocking by αPD-L1 w.t. (right); yellow arrowhead
indicates brown fat while white arrowhead points to the spleen. (B) Tracer uptake (%ID/g) for the
three imaging groups in brown fat based on quantitative region-of-interest (ROI) analysis of the
PET images. (C) Tracer uptake (%ID/g) in spleen. “*” represents P < 0.05. n = 3.
ACS Paragon Plus Environment

Page 25 of 31
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Immunofluorescent staining of brown adipose tissue for PD-L1 expression (red), and
spleen tissue for PD-L1 (cyan) and CD45 (green) expressions. Nuclei (blue) were stained as
controls.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 31
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
ACS Paragon Plus Environment

Page 27 of 31
Molecular Pharmaceutics
A.
V V
H L
N
N
1
2
3
4
5
6
7
8
N
H
H
N
V V
H L
O
H
O
C C
H L
1) In silico
screening
O
4
OH
N N N
9
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C C
H L
HO
O
2) Site-directed
mutagenesis
N
N
PBS, pH 7.2
HO
O
HC K129X
5
B.
ELISA of Fab variants’ binding to PD-L1
V V
H L
N
N
N
H
H
N
O
H
C C
H L
Wt
HC K129X
O
O
Radiolabeling
31
32
33
34
35
36
37
38
39
40
41
42
O
N
O
64Cu
5
N
N
O
O
O
ACS Paragon Plus Environment
6

Molecular Pharmaceutics
Page 28 of 31
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
ACS Paragon Plus Environment

Page 29 of 31
Molecular Pharmaceutics
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
ACS Paragon Plus Environment