Targeting protumoral tumor-associated macrophages with nanobody

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Targeting protumoral tumor-associated macrophages with nanobody-functionalized nanogels through SPAAC ligation Lutz Nuhn, Evangelin Boli, Sam Massa, Isabel Vandenberghe, Kiavash Movahedi, Bart Devreese, Jo Van Ginderachter, and Bruno G. De Geest Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00319 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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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.

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Bioconjugate Chemistry

Targeting protumoral tumor-associated macrophages with nanobody-functionalized nanogels through SPAAC ligation Lutz Nuhn,1,2,3,$ Evangelia Bolli,4,5,$ Sam Massa, 4,5 Isabel Vandenberghe,6 Kiavash Movahedi, 4,5

1

Bart Devreese,6 Jo A. Van Ginderachter, 4,5* Bruno G. De Geest1,2*

Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium

2

Cancer Research Institute Ghent (CRIG), Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium 3

Max-Planck-Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

4

Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Pleinlaan 2, 1050 Brussels, Belgium

5

Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium 6

Department of Biochemistry and Microbiology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium $

Both authors contributed equally.

KEYWORDS nanobody; nanogel; tumor-associated macrophages; click chemistry; bioconjugation.

ACS Paragon Plus Environment

1

Bioconjugate Chemistry 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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ABSTRACT

Tumor-associated macrophages (TAMs) with high expression levels of the Macrophage Mannose Receptor (MMR, CD206) exhibit a strong angiogenic and immune suppressive activity. Thus, they are a highly attractive target in cancer immunotherapy, with the aim to modulate their protumoral behavior. Here, we introduce polymer nanogels as potential drug nanocarriers which were site-specifically decorated with a Nanobody (Nb) specific for the MMR. Using azide-functionalized RAFT chain transfer agents they provide access to amphiphilic reactive ester block copolymers that self-assemble into micelles and are afterwards core-crosslinked towards fully hydrophilic nanogels with terminal azide groups on their surface. MMR-targeting Nb can site-selectively be functionalized with one single cyclooctyne moiety by maleimide-cysteine chemistry under mildly reducing conditions which enables successful chemoorthogonal conjugation to the nanogels. The resulting Nbfunctionalized nanogels were highly efficient in targeting MMR-expressing cells and TAMs both in vitro and in vivo. We believe that these findings pave the road for targeted eradication or modulation of pro-tumoral MMRhigh TAMs.

TOC GRAPHIC


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Besides cancer cells, tumors contain a vast array of non-cancerous cell types, which have been largely overlooked as potential therapeutic targets and have been labeled “the forgotten half of the tumor”. Indeed, tumors should be viewed as organoid structures in which a complex interaction takes place between transformed and non-transformed stromal cells, resulting in either tumor progression or inhibition. It is becoming clearer that therapies targeting stromal cells, such as those governing chronic cancer-associated inflammation in solid tumors, may hold new possibilities for more effective anti-cancer treatments.1 Macrophages are well known tumor-driving inflammatory cells through several mechanisms. Recent findings from our group and others have uncovered the existence of several macrophage subtypes in the tumor microenvironment.2 In this respect, tumor-associated macrophages (TAMs) with a high expression of the Macrophage Mannose Receptor (MMR, CD206) were shown to reside in hypoxic regions of the tumor or, following chemotherapy, along blood vessels. These TAMs exhibit strong angiogenic and immune suppressive activity, characteristics regulated by the hypoxic microenvironment and suggesting a strong protumoral activity.3 TAMs were also reported to contribute to diminished therapeutic responsiveness

and

tumor

relapse

after

radiation,

anti-angiogenic

therapy

and

chemotherapy in preclinical tumor models.4 Based on these convincing preclinical data, we consider MMRhigh TAMs as potential new targets for cancer therapy of nanomedicines.5 Since other TAM subsets may actually exert anti-tumoral functions, it seems a plausible approach to specifically deplete or alter MMRhigh TAM, while leaving other TAM populations untouched. In this work, we report on a strategy to functionalize polymer nanogels as potential drug nanocarriers6–8 with a Nanobody (Nb) specific for the MMR.9 Nbs are the smallest available antigen binding fragment (ca. 15kDa, 10 times smaller than a conventional antibody) derived from Camelid heavy chain-only antibodies.10,11 They lack the Fc part, hence, avoiding Fc-mediated aspecific binding found for antibody-functionalized nanoparticles.12 In


3


Page 4 of 36

addition, compared to antibody fragments (Fab), Nbs are single protein chains that can be produced recombinantly and, furthermore, engineered towards site-selective chemical modification.13 The latter allows the introduction of chemoselective linkers instead of nonspecific conjugation14,15 that might potentially interfere with binding properties of the Nb.16– 18

Importantly, anti-MMR Nbs have already successfully been exploited for theragnostic purposes9,19,20 as attractive alternative to full antibodies.21–23 However, their potential to target nanoparticles to TAMs has not yet been investigated deeply. Nanoparticle delivery to MMR+ cells has been reported by surface decoration with mannose moieties,24–27 yet this would also promote nanoparticle affinity to other mannose binding receptors, including DCSIGN, L-SIGN, Endo180, Langerin or mannose binding lectins (MBLs).28 Anti-MMR-Nbs, however, exclusively target MMR and can be genetically engineered to allow for siteselective chemical conjugation to nanoparticles without interfering with the MMR-binding site. Hence, we demonstrate in this paper a versatile approach to conjugate anti-MMR Nbs to polymeric nanogels for effective nanoparticle delivery to MMR-expressing TAMs in vitro, ex vivo and in vivo in tumor bearing mice. To conjugate Nbs to polymer nanogels, we selected the strain promoted azide alkyne cycloaddition (SPAAC), i.e. copper-free click-chemistry.29,30 Hereto, cyclooctyne-modified Nbs are first conjugated to azides located at the dangling polymer chain ends of polymerbased nanogels (Scheme 1). SPAAC is highly efficient in terms of kinetics and selectivity. The latter contributes to its popularity for bio-conjugation purposes as it is largely inert towards

functional

groups

that

occur

in

natural

amino

acids,

nucleotides

and

carbohydrates.31 Moreover, this approach also prevents unfavorable side reactions that could occur during the conjugation process, e.g. by targeting free cysteines on the nanobody

with

maleimide-functionalized

nanoparticles

(possibly

leading

to

cysteine

dimerization). From the range of available cyclooctyne derivatives for selective SPAAC, we


4

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focus in this paper on the use of dibenzocyclooctyne (DBCO) due to its excellent balance between reactivity towards azides and orthogonality towards other nucleophiles present in proteins such as amines, thiols and alcohols.32–34 As building block for nanogel fabrication we elaborate on a method introduced by the Zentel group, based on self-assembly of amphiphilic block copolymers composed of a reactive pentafluorophenyl (PFP) ester based hydrophobic polymer block.35–38 This strategy has been widely used by us27,39–41 and others42–44 to drive self-assembly of block copolymers due to solvophobic interaction between PFP-containing repeating units followed by covalent crosslinking

and

core-modification

with

drug

compounds

and/or

fluorescent

tracer

molecules. In this study, we now focus on a chemoselective nanogel corona-decoration with an anti-MMR nanobody and demonstrate a MMRhigh TAM-specific delivery inside the tumor micromilieu.

SPAAC ligation

anti-MMR nanobody decorated nanogel

azide containing nanogel

DBCO-modified nanobody

CD206/ MMR

TAM


5


Page 6 of 36

SCHEME 1. Schematic representation of targeting the MMR on TAMs with nanobody-functionalized polymeric nanogels.


6

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RESUTS AND DISCUSSION

Synthesis of azide-functionalized polymer nanogels Amphiphilic reactive ester block copolymers were synthesized by RAFT polymerization of methoxy triethyeneglycol methacrylate (mTEGMA) and pentafluorophenyl methacrylae (PFPMA) using a dithiobenzoate-based chain transfer agent functionalized with an azide (N3) moiety (Scheme 2).45 The targeted block copolymer composition was based on earlier work that elucidated the optimal block length and ratio of both blocks that yield stable sub 100 nm nanoparticles.35 Polymer characteristics are listed in Table 1. NMR and SEC experimental data are shown in Supporting Information (Figure S1-S5). The obtained block copolymers were self-assembled into micellar nanoparticles in DMSO driven by the solvophobic nature of the poly(PFPMA) block.35 Dynamic light scattering (DLS) analysis (Table 1) indicated a micellar particle size around 40 nm. TABLE 1. Properties of the precursor block copolymer used for azide endgroup modified nanogels. mono : conversion azide-CTA :

Mcalcb

t [h] [%]a

[g∙mol-1]

Mnc [g∙mol-1]

ᴆ

c

sized (DMSO) [nm]

sizee (H2O) [nm]

AIBN

N3-P(mTEGMA)12

15 : 1 : 0.1

12

80

3,300b1

3,200

1.21

n.d.

n.d.

N3-P(mTEGMA)12-bP(PFPMA)40

50 : 1 : 0.1

40

66

13,400b2

13,300

1.21

37.8 ± 1.1

51.2 ± 0.5

a: Determined by 1H-NMR (400 MHz) of the reaction mixture in chloroform-d. b1: Calculated based on the conversion and molecular weight of monomer and CTA. b2: Calculated based on the ratio of the two different blocks. c: Determined by SEC (THF) calibrated with poly(styrene) standards. d: Determined as Z-average by DLS (using cumulant analysis) of the precursor polymer at 10 mg/mL in DMSO (compare Fig. 3B). e: Determined as Z-average by DLS (using cumulant analysis) of non-degradable crosslinked nanogels measured at 0.5 mg/mL in PBS (compare Fig. 3B).

Subsequently, the nanoparticle core was functionalized with a fluorescent probe through amide bond formation between the reactive PFP esters and the primary amine of Oregon Green cadaverine, followed by crosslinking via stoichiometric treatment of one equivalent


7


PFP

ester

with

half

an

equivalent

of

the

Page 8 of 36

bisamine

crosslinker

2,2′-

(ethylenedioxy)bis(ethylamine). Afterwards, all unreacted residual PFP esters could be converted into hydrophilic repeating units by addition of an excess of 2-hydroxyethylamine, yielding fully hydrophilic nanogels after dialysis against water. DLS analyses of the nanogels shown in Figure S6 (and Figure 3B) as well as TEM images presented in Figure S7 confirmed conserved sizes of roughly 50 nm for the core-crosslinked micelles (see also Table 1), while UVvis spectroscopy demonstrated covalent conjugation of the fluorescent dye Oregon Green cadaverine (Figure S8). These data are in accordance with our earlier reports for different types of core-functionalized nanoparticles35,39,41,44,46 with different types of functional end-groups at the poly(mTEGMA) chain ends,40,42 which can be attributed to the robustness of the nanogel design approach based on the solvophobic properties of PFPactivated esters to drive self-assembly followed by core-modification. The accessibility of the azide moieties towards strain-promoted copper-free click reaction was tested by incubation with a cyclooctyne-modified fluorescent dye in PBS (see Supporting Information Scheme S3 and Figure S9). Following extensive dialysis in parallel with nanogels that were incubated with the same fluorescent dye but without the cyclooctyne functionality, only the azide-reactive cyclooctyne-dyes were detected on the nanogels (Figure S10-12). Note that this conjugation reaction did not affect the particle size distribution (Figure S13).


8

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A

2-mercaptoethylamine-HCl, PBS, 5mM Na2EDTA pH 6.5 16 h

(

)3

50mM HEPES, 5mM Na2EDTA pH 6.5 16 h

3

B

C

SCHEME 2. Chemical synthesis of (A) DBCO modification of nanobody by site-selective thiolmaleimide ligation of the N-terminal cysteine (after careful reduction of nanobody dimers without cleaving their internal disulfide bridges). (B) RAFT block copolymerization of PFPMA and mTEGMA using an azide-functional CTA followed by end-group removal by treatment with an excess of azoinitiator. (C) Nanogel assembly.

Site-selective modification of nanobodies with a cyclooctyne Next, we aimed to endow anti-MRR nanobodies (Nb) with a cyclooctyne moiety to become reactive towards triazole formation with an azide by SPAAC. To allow for site-selective introduction of a cyclooctyne moiety, anti-MMR Nbs were engineered by recombinant route with a terminal cysteine moiety. This cysteine moiety was used for the conjugation of a DBCO moiety. We preferred this route because of the fast and robust chemoselective


9


Page 10 of 36

reaction conditions that can afterwards be applied for the conjugation to the nanogel, compared to other ligation strategies. To attach the DBCO group to the terminal cysteine, a commercially available DBCO-tetraethyleneglycol-maleimide (DBCO-(PEG)4-MAL) linker was used. Due to C-terminal oxidation of cysteines, disulfide reduction prior to thioether formation between the maleimide and cysteine was required. This was initially attempted under standard TCEP-mediated reducing conditions used for this type of reaction. Whereas we found this to be effective in terms of conjugation, we also found that under these reducing conditions, the anti-MMR Nb lost its target binding properties. We attribute this to cleavage of the internal disulfide bond in the Nb, which is crucial for its bioactivity (data not shown). To

circumvent

this

issue,

we

opted

for

milder

reducing

conditions

using

2-

mercaptoethylamine hydrochloride (2-MEA) at a concentration of 10 mM, which is capable of reducing disulfide-based dimer formation between single Nbs, but which does not affect the internal disulfide bond,47 probably due to limited solvent accessibility for the positively charged reducing agent. The reaction was performed at a 300:1 molar ratio of 2-MEA to Nb. Overnight reduction proved to be sufficient to yield quasi full conversion of dimer to monomeric Nb (Figure 1A and Figure S14). Subsequently, reduced Nb monomers were isolated by a desalting column (Figure S15) and immediately treated with 50 equivalents of (DBCO-(PEG)4-MAL). Afterwards, the resulting Nb-DBCO conjugates were purified by fast protein liquid chromatography (FPLC) (Figure 1B1 and Figure S16) and analyzed by UVvis spectroscopy, SDS-PAGE, MALDI-TOF and ESI-MS. Interestingly, the presence of the DBCO moiety induces a slight shift in the FPLC trace which allows to discriminate between DBCO-modified and unmodified Nb, as indicated by UV-Vis analysis of the respective fractions that were collected and combined accordingly (Figure 1B2 and Figure S17). SDS-PAGE depicts a single band of DBCO-Nb at about half the retardation length of the dimer Nb starting product (Figure 1C and Figure S18, the reduced nanobody starts to


10

Page 11 of 36

dimerize over time again). The gain in exact molecular mass measured by MALDI-TOF and ESI-MS analysis (Figure S19-S21) confirmed the presence of a single DBCO moiety on the DBCO-Nb fraction isolated by FPLC (Figure 1D – note that the isolated Nb fraction which could not be modified by DBCO-(PEG)4-MAL had formed a C-terminal disulfide with 2-MEA,

S

H N

O OH

absorbance @ 215 nm [a.u.]

SH

16h

50 kDa

60

37 kDa

40

25 kDa 20 kDa 15 kDa

20

10 kDa

0 10

B2

15

D

20

elution volume /mL

2-MEA reduced Nb

2-MEA reduced Nb

O OH S S

HO O

N H

intensity [a.u.]

S

H N

absorbance / a.u.

absorbance / a.u.

S

DBCO-Nb

100

DBCO-Nb

2h H 2N

dimer

reduced Nb DBCO-Nb (reaction mixture)

80

absorbance @ 280 nm [a.u.]

S

C

100

during reduction

B1 H2N

ladder

A

purified DBCO-Nb

see Scheme S5).

S S

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


75

50

NH 2

25

0h 8

10

12

14

16 250

elution volume/ mL

300 350 wavelengt / nm

400

250

300 350 wavelengt / nm

400

0 14000

15000 16000 m/z [g/mol]

17000

FIGURE 1. (A) FPLC trace of nanobody dimer before, during and after reduction with 2mercaptoethylamine hydrochloride (2-MEA). (B1) FPLC trace overlay of reduced nanobody and the reaction mixture after conjugation to DBCO-(PEG)4-MAL and (B2) resulting UV-vis spectra of the isolated 2-MEA reduced nanobody and the desired DBCO-nanobody (theoretically calculated spectra plotted with dotted lines). (C) SDS-PAGE of the isolated DBCO-nanobody, the reduced nanobody before modification with DBCO and its dimer form. (D) MALDI-TOF spectra of the isolated DBCOnanobody and 2-MEA reduced nanobody.

To investigate whether DBCO-modified Nb can still bind to its MMR target receptor, we conjugated a fluorescent azide-functional dye (i.e. BDP-FL azide, an azide-functionalized bodipy analogue) to the DBCO-Nb by SPAAC (Scheme S6). Extensive dialysis was performed to remove unbound dye (in parallel dye without DBCO-modified Nb was dialyzed


11


as well, to monitor release of the dye from the dialysis tube). Afterwards, the presence of the covalently conjugated dye to protein could be visualized optically (Figure S22) and was further confirmed by UV-Vis spectroscopy (Figure S23). Target-binding experiments of the modified nanobody were performed on Chinese hamster ovarian (CHO) cells which were genetically engineered to express the MMR (CHOMMR+). As negative control, CHO cells were used that were negative for the MMR (CHOMMR-). Presence and absence of the MMR was confirmed by FACS analysis of cells stained with a commercial anti-MMR (i.e. anti-CD206) antibody (Figure 2). Incubation of BDP-FL labeled DBCO-nanobody revealed clear binding to receptor positive CHOMMR+ and minimal binding to receptor negative CHOMMR- (Figure 2). We attribute the slight binding to CHOMMR- cells to the hydrophobic interaction of the BDP label that was introduced via an aliphatic linker onto the hydrophobic DBCO group with the phospholipid cell membrane, since the specificity of the Nb for MMR has been unequivocally demonstrated in our previous study.9

A

CHOMMR-

◊ % of max

% of max

CHOMMR-

CHOMMR+ % of max

% of max

CHOMMR+

CD206

Nb 25000

9000 7500

MFI BDP FL [a.u.]

B MFI AF647 [a.u.]

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

Page 12 of 36

6000 4500 3000 1500 0

20000 15000 10000 5000 0

MMR+ CHO

MMRCHO

antibody PBS

MMRCHO

MMRCHO

nanobody PBS


12

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FIGURE 2. (A) Flow cytometry analysis of MMR-receptor positive and negative CHO cells pulsed with Alexa Fluor 647-labeled anti-MMR antibody and (SPAAC) BDP FL-labeled anti-MMR nanobody: histograms (A) and corresponding mean fluorescence intensity (MFI) values (B).

Synthesis of anti-MMR Nb-nanogels For conjugation of DBCO-Nb to N3- functionalized nanogels, DBCO-Nb N3-nanogels were first reacted at increasing molar ratios of azide on the nanogels to DBCO-Nb for 16 h at room temperature. Afterwards, SDS-PAGE analysis showed that saturation occurs roughly at an azide-nanogel : DBCO-Nb ratio of 30:1 (Figure 3A and Figures S24 – note that a quantification error of conjugation efficiency of at least 10% can be considered due to the staining procedure/ optical camera imaging). DLS analysis of the samples showed no changes in colloidal stability or size distribution upon conjugation of Nb (Figure 3B and Figure S25).

To test whether Nb-nanogels can be internalized by MMR-expressing cells, CHOMMR+ and CHOMMR-

cells were incubated

with the same concentrations (confirmed

by

UVvis

spectroscopy, see Figure S26) of Oregon Green labeled Nb-modified and blank nanogels, respectively, followed by FACS analysis (gating strategy shown in Figure S27). These experiments (Figure 4) show a clear difference in nanogel uptake by MMR+ cells versus MMR- cells, whereby CHOMMR+ cells showed a strong uptake of Nb-nanogels, but not blank nanogels, whereas this Nb-driven uptake was largely absent in the case of CHOMMR- cells. Confocal microscopy confirmed that the Nb-nanogels became internalized by CHOMMR+ cells and did not merely bind to the cell surface. This suggests the potential to exploit MMR as an internalizing receptor for the targeted intracellular delivery of bioactive compounds.


13

A1

Page 14 of 36

azide : DBCO-Nb ratio 15:1

30:1

50:1

75:1

100:1

250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa

A2

% conjuation efficiency

10 kDa

100 80 60 40 20 0

0:1

15:1

B

30:1 50:1 ratio

75:1

100:1

Nb-nanogel

% volume

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

DBCO-Nb


nanogel

N3-P(mTEGMA)12 -b-P(PFPMA)40 (in DMSO)

1

10

100

1000

size [nm]

FIGURE 3. Characterization of SPAAC-mediated nanobody conjugation. (A) SDS-PAGE of DBCO-modified nanobody with increasing molar ratios of N3-nanogel to nanobody (A1) and its corresponding conjugation efficiencies (A2). Note that due to their size, nanobody-conjugated nanogels cannot penetrate the acrylamide gel well and are often washed off during the Coomassie staining procedure. (B) DLS size distribution of the nanogel particles (N3-P(mTEGMA)12-b-P(PFPMA)40 precursor polymer micelles in DMSO (grey), core-crosslinked nanogels before (red) and after (blue) conjugation of DBCO-modified nanobodies).


14

Page 15 of 36

A

CHOMMR+

C CHOMR+

Nb-nanogel phalloidin

CHOMR-

Nb-nanogel phalloidin

nanogel Nb-nanogel

% of max

PBS

CHOMMRnanogel Nb-nanogel PBS

12000

B1

nanogel Nb-nanogel PBS

10000 8000 6000 4000 2000 0 CHOMMR+

CHO MMR-

B2

% oregon green positive cells

nanogel

MFI [a.u.]

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


50

nanogel Nb-nanogel

40

PBS 30 20 10 0 CHO MMR+

CHO MMR-

FIGURE 4. In vitro evaluation of anti-MMR Nb-nanogels. (A) Flow cytometry histograms of MMRreceptor positive and negative CHO cells pulsed at 37°C with Nb-nanogels and naked nanogels. (B) Corresponding MFI values (1) and % nanogel positive cells (2). (C) Confocal microscopy images of MMR-receptor positive and negative CHO cells pulsed at 37°C with Nb-nanogels.


15


Page 16 of 36

Ex vivo evaluation of anti-MMR Nb-nanogels Next, we aimed at evaluating the potential of anti-MMR Nb functionalized nanogels in a more physiologically relevant setting. For this purpose, 3LL-R (Lewis Lung carcinoma) tumors were grown subcutaneously in mice, followed by dissection and collagenase treatment to generate a tumor single cell suspension. Of note, these tumors contain a high macrophage content (between 20-50%, depending on tumor size), which is not uncommon in clinical samples as well. This cell mixture, representing all cell types present within a tumor, was treated with Oregon Green labeled Nb-nanogels or blank nanogels, followed by FACS analysis (gating strategy shown in Figure S28). Treatment was either performed at 4°C, allowing the surface binding of the nanogels but blocking energy-dependent endocytotic uptake, or at 37°C which allows endocytosis. Of note, our previous research demonstrated the presence of MMRhigh and MMRlow TAMs in the tumor microenvironment, which can also be identified as MHC-IIlow and MHC-IIhigh TAMs, respectively.2 This differential MHC-II expression allowed us to discriminate by FACS between MMRhigh and MMRlow TAMs without the need to perform additional anti-MMR staining, which could cause competition with Nb-nanogel binding. FACS analysis upon treatment at 4°C indicated a significant increase in Nb-nanogel binding by MHC-IIlow/MMRhigh TAMs compared to MHC-IIhigh/MMRlow TAMs (Figure 5A). In addition, Nb-nanogel binding is significantly higher than naked nanogel binding on MHC-IIlow/MMRhigh TAMs, but not on MHC-IIhi/MMRlo TAMs (Figure 5A), suggesting Nb-mediated targeting of the nanogel to MMR-expressing tumor macrophages. Similar results were obtained on sorted MHC-IIlow/MMRhigh TAMs ex vivo (Figure S29). At 37°C, an overall increase in fluorescence can be found, which is most like due to enhanced energy-dependent nanoparticle internalization. Yet, a preferred cellular association in MHCIIlow/MMRhigh TAMs was found for Nb-nanogels exclusively (Figure 5B).


16

Page 17 of 36

single cell suspension

tumor dissection

tumor inoculation

Nb-nanogel pulsing & FACS analysis

A

B

1500

*

15000

*

*

Nb-nanogel nanogel

1000

10000

∆MFI

∆MFI

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


500

5000

0 MHC II LOW TAMs

MHC IIHIGH TAMs

0

MHC II LOW TAMs

MHC IIHIGH TAMs

FIGURE 5. Ex vivo evaluation of anti-MMR Nb-nanogels. Total 3LL-R tumor cell suspensions were incubated in vitro with either anti-MMR-Nb-nanogels (Oregon Green) or naked nanogels (Oregon Green) for 2h at (A) 4°C and (B) 37°C, followed by FACS analysis. ∆ MFI ± s.e.m. are indicated and represent (MFI Oregon Green-MFI PBS control), n = 3 mice. Statistical analysis was performed by the Student’s t test, p

Targeting protumoral tumor-associated macrophages with nanobody

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