Iga Mediated Killing of Tumor Cells by Neutrophils Is Enhanced by CD47-Sirpα Checkpoint Inhibition

Author Manuscript Published OnlineFirst on November 5, 2019; DOI: 10.1158/2326-6066.CIR-19-0144 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 IgA mediated killing of tumor cells by neutrophils is enhanced by CD47-SIRPα checkpoint inhibition

2 Running title: CD47-SIRP limits IgA mediated killing by neutrophils

4 Louise W. Treffers1,*, Toine Ten Broeke2,*, Thies Rösner3,*, J.H. Marco Jansen2,*, Michel van Houdt1,

5 Steffen Kahle3, Karin Schornagel1, Paul J.J.H. Verkuijlen1, Jan M. Prins6, Katka Franke1, Taco W.

6 Kuijpers1,4, Timo K. van den Berg1,5,**, Thomas Valerius3,**, Jeanette H.W. Leusen2,**, Hanke L.

7 Matlung1,**

8 1Sanquin Research, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam,

9 Amsterdam, The Netherlands

10 2Immunotherapy Laboratory, Laboratory of Translational Immunology, University Medical Center

11 Utrecht, Utrecht, The Netherlands

12 3Section for Stem Cell Transplantation and Immunotherapy, Department of Internal Medicine II,

13 Christian-Albrechts-University, Kiel, Germany

14 4Emma Children’s Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands

15 5Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit

16 Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands

17 6Department of Internal Medicine, Division of Infectious Diseases, Academic Medical Center,

18 University of Amsterdam, The Netherlands.

19 * and **, these authors contributed equally

20 Keywords: ADCC, IgA antibody, CD47-SIRPα interactions, antibody therapy, neutrophils 21 Disclosures 22 T.K. van den Berg is the inventor on the patent application WO2009/131453 A1, which is owned by 23 Sanquin and licensed to Synthon Biopharmaceuticals BV. Sanquin has a collaborative agreement with 24 Synthon Biopharmaceuticals BV, with active involvement of K. Franke, H.L. Matlung and T.K. van den 25 Berg for the development of agents targeting CD47-SIRPα in cancer. J.H.W. Leusen is an inventor on 26 several patents on IgA in therapeutic application in cancer, and is the scientific founder and 27 shareholder of TigaTx. T. Valerius is a scientific advisor for TigaTx. 28 Correspondence to: 29 Hanke L. Matlung 30 Sanquin Research 31 Plesmanlaan 125 32 1066 CX Amsterdam 33 The Netherlands

Downloaded from cancerimmunolres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 5, 2019; DOI: 10.1158/2326-6066.CIR-19-0144 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

34 Tel.: +31 20 512 3261 35 Fax: +31 20 512 3310e-mail: [email protected] 36 37 38

39 Abstract

41 Therapeutic monoclonal antibodies (mAbs), directed towards either tumor antigens or inhibitory

42 checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the

43 therapeutic efficacy of these tumor antigen-targeting mAbs is mediated - at least partially - by

44 myeloid effector cells, which are controlled by the innate immune checkpoint interaction between

45 CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRP

46 interactions can substantially potentiate antibody-dependent cellular phagocytosis (ADCP) and

47 cytotoxicity (ADCC) of tumor cells by IgG antibodies both in vivo and in vitro. IgA antibodies are

48 superior in killing cancer cells by neutrophils compared to IgG antibodies with the same variable

49 regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we

50 show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA

51 antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and

52 IgA antibodies against different antigens, i.e. HER2/neu and EGFR. Consequently, combining IgA

53 antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating

54 cancer cells in vivo. In a syngeneic in vivo model, the eradication of cancer cells was predominantly

55 mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We

56 conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα

57 checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in

58 combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor

59 patients.

62 Introduction

63 Monoclonal antibodies targeting tumor antigens are widely used and an effective treatment of

64 various malignancies. Over the years an increasing number of mAbs targeting different tumor

65 antigens have been approved for use in cancer patients including trastuzumab or cetuximab, which

66 target HER2/neu or EGFR respectively (1). All of the anti-cancer mAbs on the market are of the IgG

67 class (2), but the potential of other isotypes, such as IgA, are also being explored. IgA is known for its

68 anti-microbial role and is abundantly present at mucosal sites in its dimeric form (3). As a monomer,

69 IgA is the second most prevalent antibody in serum (3). IgA is comprised of two subclasses, IgA1 and

70 IgA2, that bind with similar affinity to the myeloid IgA receptor (FcαRI, CD89) (4). Many cancer

71 therapeutic antibodies act by a combination of both direct, as well as indirect, immune-mediated

72 effects, which include cytotoxicity induced by complement activation, antibody-dependent cellular

73 phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) (5). ADCC can be mediated

74 through activation of different Fc-receptor expressing cells, including natural killer (NK) cells, macrophages

75 and neutrophils (6-9). Of these Fc-receptor expressing effector cells, macrophages and neutrophils express

76 FcαRI that binds IgA antibodies, and hence can kill tumor cells by ADCP or ADCC as demonstrated in vitro

77 (9-12). Antibodies of IgA isotype are effective in inducing ADCC of various tumor targets, including

78 HER2/neu+- and EGFR+-carcinomas and CD20+ lymphomas (9, 13, 14). When neutrophils are used as

79 effector cells, IgA therapeutic antibodies induced significantly higher levels of ADCC compared to IgG

80 variants of the same mAb (9, 13-17).

81 Checkpoint inhibition of the inhibitory receptor signal regulatory protein alpha (SIRPα) or its ligand

82 CD47 is effective in pre-clinical models when combined with IgG1 and IgG2-based cancer therapies

83 (18-23). Based on these results CD47-SIRP interaction blocking agents are being tested in clinical

84 trials for hematological and solid cancers (www.clinicaltrials.gov identifiers: NCT02216409;

85 NCT02678338, NCT02641002; NCT02367196, NCT02890368; NCT02663518, NCT02953509)(24, 25). A

86 published phase 1b study shows encouraging results for a blocking anti-CD47 antibody in

87 combination with rituximab resulting in durable complete responses in patients with Non-Hodgkin

88 lymphoma (25). SIRPα is selectively present on myeloid cells and limits ADCC by macrophages and

89 neutrophils (18). SIRPα‘s ubiquitously expressed ligand CD47 acts as a ‘don’t eat me’ signal and was

90 found to be often overexpressed on cancer cells, inhibiting phagocytosis and clearance by

91 macrophages (20, 26-28). In a clinical setting, the expression level of CD47 on cancer cells is inversely

92 related to patient response to anti-cancer antibody therapy (18, 20, 27-29). In several pre-clinical

93 models, CD47-SIRPα blockade is a promising target for enhancing cancer immunotherapies when

94 combined with IgG mAbs targeting different tumor antigens, including cetuximab, trastuzumab and

95 rituximab (18, 20, 21). However, for IgA antibodies directed against specific tumor antigens, this

96 enhancing effect of additional CD47-SIRPα checkpoint inhibition has not yet been investigated. Here,

97 we examined whether inhibiting CD47-SIRPα interactions lead to an enhancement of IgA-based

98 cancer therapies in both in vitro systems and in vivo mouse models. We demonstrated a prominent

99 increase in IgA-opsonized tumor cell clearance in the presence of CD47-SIRP blockade, providing

100 support for the idea that targeting the CD47-SIRPα checkpoint could be used to improve IgA

101 therapeutic antibodies in cancer.

102 Materials and Methods

103 Cells and culture

104 Cell lines were from ATCC (A431, SKBR3, BT-474, RAW264.7) Ba/F3) or DSMZ (Kyse-30). Ba/F3 cells

105 were a kind gift from the group of Dr. Leo Koenderman and were re-authenticated by PCR analysis by

106 IDEXX in 2017. All other cells were kept in culture according the suppliers’ recommendations and

107 were obtained between 2011 and 2018. Cells were tested for Mycoplasma and kept in culture and

108 used for experiments for a maximum of three months.

109

110 Generation of genetically modified cells

111 A431-CD47KO cell lines were generated by lentiviral transduction of pLentiCrispR-v2 – CD47KO

112 (pLentiCrispR-v2 was a gift from Feng Zhang (Addgene plasmid #52961)); A431 cells expressing

113 HER2/neu (A431HER2/neu) were generated by retroviral transduction, followed by positive selection

114 based on puromycin resistance, as previously described (10). SKBR3-CD47KD cells were generated by

115 lentiviral transduction of pLKO.1-puro – CD47KD. Transduced and afterwards puromycin selected

116 cells showed a CD47 expression of 10-15% of normal. As control cell line a scrambled shRNA was

117 used. Knockdown and knockout of antigens on cell lines was routinely verified by flow cytometry.

118 Ba/F3 cells expressing EGFR were transfected with WT EGFR (Upstate) and EGFR expressing clones

119 were selected using neomycin. Ba/F3 cells expressing HER2/neu were generated by retroviral

120 transduction, followed by positive selection using puromycin resistance and limiting dilution.

121

122 Antibodies and reagents

123 SIRP or CD47 were blocked using human SIRP mAb 12C4 or F(ab’)2 fragments of human CD47

124 antibody B6H12 (both at 10 µg/mL), respectively (18). IgG trastuzumab (Roche), IgG cetuximab

125 (Merck KGaA), anti-HER2-IgA2and anti-EGFR-IgA2) were generated as described (15) and used at a

126 final concentration of 5 μg/mL, unless stated otherwise. To block mouse SIRPα we used the rat IgG2

127 antibody MY-1, generously gifted to us by the group of prof. dr. Takashi Matozaki (University of

128 Kobe, Japan), at a final concentration of 10 μg/mL in our in vitro studies (30).

129

130 Neutrophil isolation

131 Human neutrophils were isolated either by density centrifugation of heparinized blood over isotonic

132 Percoll (Pharmacia Uppsala, Sweden) followed by red cell lysis with hypotonic ammonium chloride

133 solution at 4°C (31) or by using PolyMorph Prep® (catalogue number 1114683, ProGen). After

134 isolation, neutrophils were cultured in HEPES+ medium (containing 132mM NaCl, 6.0 mM KCl, 1.0mM

135 CaCl2, 1.0 mM MgSO4, 1.2 mM K2HPO4, 20 mM Hepes, 5.5 mM glucose and 0.5% HSA) and

136 immediately used at a concentration of 5x106 cells/mL, unless stated otherwise, or stimulated for 30

137 minutes at 37°C with 50 U/mL GM-CSF (CellGenix) before use. All blood was obtained from healthy

138 individuals after written informed consent. Studies on human blood samples were conducted after

139 written informed consent and according to the Declaration of Helsinki 1964. Blood from healthy

140 volunteers was collected after approval by the Sanquin Research institutional medical ethical

141 committee, in accordance with the standards laid down in the 1964 Declaration of Helsinki.

142 Two FHL5 patients were used in this study; for patient characteristics see patients B and C described

143 in (32). Four individual CGD patients were used in this study, 2 male patients with mutations in the

144 gp91 gene CYBB and 2 female patients with mutations in the p47phox gene NCF1.

145 Mouse neutrophils were isolated from mouse bone marrow as follows: Femurs were crushed in a

146 mortar, washed with MACS buffer (containing phosphate buffered saline (PBS)) and filtered through

147 a 40 µm filter. Rat anti mouse-CD16/CD32 (BD Pharmingen, clone 2.4G2) was incubated in 1 mL

148 MACS-buffer, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA) at a concentration of 25

149 μg/mL for 20 minutes on ice. After incubation, anti-Ly6G-APC (clone 1A8, BD Biosciences) was

150 directly added at a concentration of 1 μg/mL for 30-45 minutes on ice. After washing the cells in

151 MACS buffer, finally, 20 μL anti-APC MicroBeads (Miltenyi Biotec) were added per 106 cells for 60

152 minutes on ice. After isolation, mouse neutrophils were cultured overnight in RPMI supplemented

153 with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml

154 streptomycin at a concentration of 5*106 cells/mL, in the presence of 50 ng/mL mouse IFNγ

155 (PeproTech) and 10 ng/mL clinical grade G-CSF (Neupogen), after which the cells were used in assays

156 the following day.

157 ADCC

158 Cancer cell lines were labeled with 100 μCi 51Cr (Perkin-Elmer) for 90-120 minutes at 37°C. After 3

159 washes with PBS, 5x103 cancer cells were co-incubated with neutrophils (see figure legends for E:T

160 ratios) in 96-well U-bottom plates. The co-culture was in IMDM cell culture medium (SKBR3, BT-474)

161 or RPMI culture medium (A431, Kyse-30, Ba/F3) supplemented with 20% or 10% (v/v) FCS

162 respectively and 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin for 3-4 hours at

163 37°C and 5% CO2 and contained therapeutic antibodies against HER2/neu or EGFR (see figure legends

164 for concentrations used). After incubation, the supernatant was harvested and analyzed for

165 radioactivity using a gamma counter (Wallac). The percentage of cytotoxicity was calculated as

166 [(experimental cpm- spontaneous cpm)/ (total cpm- spontaneous cpm)] x 100%. All conditions were

167 measured in triplicate.

168 Trogocytosis assay

169 To determine tumor membrane uptake by neutrophils, a FACS based assay was used. Cancer cells

170 were labeled with a lipophilic membrane dye (DiO/DiD, 5µM, Invitrogen) for 30 minutes at 37°C.

171 After washing the target cells with PBS they were incubated with neutrophils in a U-bottom 96-well

172 plate at a 5:1 E:T ratio in the absence or presence of 5μg/mL therapeutic antibodies against

173 HER2/neu or EGFR. Samples were fixed with stopbuffer containing 0.5% PFA, 1% BSA and 20 mM NaF

174 and measured by flow cytometry as described below. After gating for the neutrophil population, the

175 mean fluorescent intensity (MFI) of cells positive for DiO/DiD were determined.

176 Live cell imaging

177 Target cells were cultured on glass coverslips (25 mm or 30 mm diameter) and their membranes

178 were labeled with DiD according to manufacturers’instructions (5 µM, catalogue number V22887

179 Invitrogen). Target cells were incubated with neutrophils in a 5:1 E:T ratio for time periods up to 4

180 hours at 37°C and 5% CO2 in IMDM culture medium (Gibco) supplemented with 20% FCS. Imaging

181 was started within 5 minutes after initiation of the experiment and was performed at the indicated

182 times and intervals using a Leica TCM sp8 confocal microscope (Leica).

183 Mouse models

184 Experiments for the Ba/F3 peritoneal model (discussed below) were performed with 12- to 38-wks

185 old male and female human FcαR transgenic (Tg) mice which were generated at UMC Utrecht (33)

186 and backcrossed on a Balb/cByJRj background (Janvier, France) and maintained in hemizygous

187 breeding. Transgene negative (NTg) littermates were used as control mice. Experiments for the A431

188 in vivo model (discussed below) were performed with 17- to 70-wks old female human FcαR

189 transgenic (Tg) mice and backcrossed on a SCID background (CB17/lCR-PrkdcSCID/Crl, Charles River)

190 and maintained in hemizygous breeding. Mice on SCID background were used to allow engraftment

191 of A431 cells and has the advantage to not exaggerate the role of CD47-SIRPα interactions as is the

192 case in i.e. NOD SCID mice in which NOD SIRPα exhibits a ~50 times higher affinity for human CD47

193 (34). All mice were bred in the specific pathogen-free facility of the Central Animal Laboratory of

194 Utrecht University, all animal experiments were conducted in accordance with, and with the

195 approval of the Animal Ethical Committee of the UMC Utrecht (license# AVD115002016410).

196 The Ba/F3 peritoneal model was described previously (9). Briefly, Ba/F3-HER2 and Ba/F3 cells were

197 labelled with respectively 2 µM or 10 µM CT violet (Invitrogen, Thermofisher) for 15 min at room

198 temperature and mixed thereafter at 1:1 ratio. In total 1x107 cells were injected per mouse

199 intraperitoneally (i.p.) in 200 μL PBS.

200 200 μg My-1 was used to block mouse SIRPα in vivo which was injected 2 days before tumor

201 cell injection and mixed with the treatment consisting of anti-HER2-IgG1 or anti-HER2-IgA2 (100 μg)

202 injected i.p. directly after the injection of tumor cells. Antibodies were diluted in PBS and injected in

203 a volume of 200 µl. Sixteen hours later, these mice were euthanized, the peritoneum washed with

204 PBS containing 5mM EDTA, the absolute number of Ba/F3-HER2 and Ba/F3 determined by flow

205 cytometry using TruCount tubes (BD biosciences) and the ratio of Ba/F3-HER2 and Ba/F3 was

206 calculated. Effector cells in the peritoneum were determined using specific antibodies (described

207 below) and their relative amount was normalized to a constant amount of beads (Invitrogen). Gating

208 strategies for defining the different subpopulations are depicted in Supplemental Fig. S1.

209 For the A431 in vivo model mice were injected with 5x105 A431-CD47KO cells on the right

210 flank and 5x105 A431 scrambled (A431-SCR) control cells were injected in the same mouse on the left

211 flank. On day 6 all the mice had palpable A431-CD47KO and A431-SCR control tumors,

212 thusintravenous (i.v.) treatment via the tail vein was started with a single injection of 50 μg IgG

213 cetuximab or 250 μg anti-EGFR-IgA2. Antibodies were diluted in PBS and injected in a volume of 100

214 μl. Anti-EGFR-IgA2 has a shorter half-life compared to cetuximab, therefore anti-EGFR-IgA2

215 treatment was continued by i.p. injections on days 8, 10, 13, 15 and 17 (250 ug). Tumor outgrowth

216 was measured twice a week with calipers and volume was calculated as length x width x height.

217 Bead binding assay

218 The SIRPα expressing murine macrophage cell line RAW 264.7 was used to show the blocking

219 capabilities of the mouse anti-SIRPα antibody MY-1, using a bead binding assay. In short: 105 RAW

220 264.7 cells were seeded in a V shape 96 well plate and washed with PBS containing 0.1% BSA. Cells

221 were then incubated in PBS containing 0.1% BSA and 10 μg/mL anti-SIRPα blocking antibody (MY-1)

222 and kept on ice for 30 minutes. Another vial containing 1 μg/mL mouse CD47-Fc together with

223 20μg/mL goat anti-human Alexa647 and 1% normal rat serum was incubated on ice for 30 minutes.

224 Cells were washed with PBS containing 0.1% BSA, after which 50 μL mouse CD47 goat anti-human

225 Alexa647-NRS complex was added to cells and kept on ice for 30 minutes. Finally, cells were washed

226 in HEPES+ buffer and binding was determined using flow cytometry as described below.

227 Flow cytometry

228 Effector cells in the peritoneum were determined after incubation with PBS containing 5% normal

229 mouse serum (Equitech-bio) for 45 min at 4-7oC. Subsequently, the following fluorescently labelled

230 antibodies were used for 45-60 min at 4-7oC to stain for different effector cells types: B220 (RA3-6B2)

231 C, I-A/ I-E (M5/114.15.2), CD8 (53-6.7), Ly-6G (1A8), CD45 (30-F11), CD4 (RM4-5), F4/80 (BM8), all

232 fromBiolegend, and CD11b (m1/70, BD biosciences).

233 After excluding Ba/F3 cells from the analysis based on their CT violet staining, granulocytes were

234 identified as Ly-6G+/ CD11b+ and F4/80-, macrophages were identified as F4/80+/ CD11b+, and Ly-

235 6G-lymphocytes were analyzed by first excluding Ba/F3 cells, F4/80+/ CD45+ macrophages and dead

236 cells (7AAD+) followed by CD45 selection where B cells were identified as B220+/ I-A/ I-E+ and T cells

237 as being CD4+ or CD8a+. Saturation of SIRPα in vivo was determined by comparing staining for the

238 injected MY-1 with anti-rat Ig (BD biosciences) with ex vivo added MY-1 or isotype control and anti-

239 rat Ig both followed by staining for macrophages and granulocytes. Measurements were performed

240 on a FACSCantoII (BD biosciences), data were analyzed using FACS Diva software (BD biosciences)

241 For the CD47-bead binding assay, goat anti-human Alexa647 IgG (H+L) (Invitrogen) was used at a

242 concentration of 20 μg/mL.

243 Cell lines were analyzed for expression of HER2 using trastuzumab, EGFR using cetuximab, human

244 CD47 using B6H12 (18), murine CD47 using miap301 (eBioscience), murine SIRPα using MY-1 (30).

245 CD20 and CD47 expression of Raji cells were characterized using CD20-FITC (Beckmann Coulter) or

246 CD47-PE (Miltenyi Biotec), respectively.

247 Data analysis and statistics

248 Statistical differences between two groups were tested using (paired) t test; multiple comparisons

249 were tested using two-way ANOVA with Tukey’s correction for multiple tests or one-way ANOVA-test

250 followed by Sidak or Dunett post-hoc test for correction of multiple comparison by GraphPad Prism

251 (GraphPad Software, version 7 and 8). A P value less than 0.05 was considered as statistically

252 significant.

253

254 Results

255 IgA-mediated ADCC was restricted by CD47-SIRPα interactions

256 IgA mediated ADCC by neutrophils is superior to IgG mediated ADCC (9-11). However, it is still

257 unknown whether this is also controlled by CD47-SIRPα checkpoint inhibition as is seen with IgG

258 antibodies (10-12). This is particularly relevant as IgA mediated killing is induced by a unique FcR, i.e.

259 FcRI (CD89), not by FcR. Inhibition of CD47-SIRPα interactions significantly increases IgG-mediated

260 phagocytosis of cancer cells by macrophages and killing by stimulated neutrophils, whilst neutrophils

261 are normally quite inefficient in performing IgG-mediated ADCC of cancer cells (18, 20, 21). To

262 investigate the role of SIRPα as a checkpoint in IgA-mediated ADCC by freshly isolated neutrophils,

263 we used the HER2/neu positive breast cancer cell line SKBR3 and the EGFR positive epidermoid

264 carcinoma cell line A431 (Fig. 1A). Furthermore, we verified CD47 expression across the two different

265 cell lines (Fig. 1A). Disruption of CD47-SIRPα interactions was achieved by either genetically

266 decreasing CD47 expression (CD47KD) on SKBR3 or by genetically deleting CD47 expression (CD47KO)

267 on A431 cells (Fig. 1A), without interfering with HER2/neu or EGFR (Fig. 1A). In 51Cr-release assays,

268 the different IgA2 antibodies targeting HER2/neu or EGFR significantly increased neutrophil ADCC

269 towards the opsonized target cells compared to their respective IgG antibodies (Fig. 1B-C).

270 Downregulation of CD47 by either CD47KD or by CD47KO led to significant increases in IgA mediated

271 ADCC by freshly isolated neutrophils compared to unmodified target cells (Fig. 1B-C). Even at lower

272 effector:target ratios (below 25:1) this enhancement in neutrophil-mediated killing of antibody-

273 opsonized A431 cells was observed for both IgG and IgA antibodies when deleting CD47-SIRPα

274 interactions (Fig. S2A). Stimulation of neutrophils by GM-CSF dose-dependently increased their

275 potency to perform ADCC with either IgG or IgA therapeutic antibodies in the absence or presence of

276 CD47 on the tumor cells (Fig. S2B). Experiments shown in Figure 1 demonstrated the importance of

277 CD47-SIRPα interactions on IgA mediated neutrophil ADCC by genetically interfering with CD47

278 expression on tumor cells.

279 Next, we wanted to investigate the effect of directly blocking CD47-SIRPα interactions. We

280 compared IgA mediated killing by neutrophils against wildtype and CD47KD SKBR3 or CD47KO A431

281 cells (Fig. 2A) and with the addition of either CD47 (Fig. 2B) or SIRPα (Fig. 2C) blocking antibodies.

282 Both the blockade of CD47 or SIRPα by genetic disruption or antibodies resulted in significantly

283 enhanced neutrophil-mediated ADCC by IgA antibodies (Fig. 2A-C). Thus, enhancement of neutrophil

284 ADCC was independent of the method targeting the CD47-SIRPα axis: CD47-SIRPα checkpoint

285 inhibition by CD47 or SIRPα antibodies significantly increased neutrophil mediated ADCC of

286 additional target cells such as EGFR+ Kyse-30 and HER2/neu+ BT-474 cells by IgA2 variants of

287 cetuximab and trastuzumab, respectively (Fig S2C-D). Together, these results showed that the ADCC

288 capacity of IgA antibodies is enhanced by CD47-SIRPα checkpoint blockade, similar to ADCC mediated

289 by IgG therapeutic antibodies.

290

291 Trogocytosis by neutrophils of IgA-opsonized cancer cells

292 The cytotoxic mechanism by which neutrophils perform ADCC against cancer cells has not been well

293 characterized. Recently, we described that IgG-mediated neutrophil ADCC involves a unique cytotoxic

294 process, termed trogoptosis, and that trogocytosis (the acquisition of target cell plasma membrane

295 fragments by neutrophils) is instrumental in this trogoptotic killing mechanism (32). To investigate

296 whether IgA-opsonization of cancer cells also induced trogocytosis, as described for IgG (29), we used

297 live cell imaging. IgA antibody-opsonized cancer cells were labeled with a fluorescent membrane dye,

298 and neutrophils were added as effector cells. We detected neutrophils taking up pieces of IgA-

299 opsonized cancer cell membrane (Fig. 3A-F), similar to IgG opsonized cancer cells. This was further

300 quantified using a FACS based trogocytosis assay, which showed enhanced trogocytosis of anti-HER2-

301 IgA2 opsonized breast cancer cells compared to the trastuzumab control (Fig. 3G). CD47-SIRPα

302 checkpoint inhibition led to a significant increase in neutrophil-mediated trogocytosis compared to

303 anti-HER2-IgA2 alone (Fig. 3G Fig. S2E).

304 We describe that IgG mediated ADCC by neutrophils is not dependent on their classical anti-microbial

305 effector mechanisms, such as granule exocytosis and NADPH oxidase activity (32, 35, 36). To rule out

306 the contribution of these anti-microbial effector functions in IgA mediated tumor cell destruction, as

307 seen with IgG (29), we made use of neutrophils from rare familial hemophagocytic

308 lymphohistiocytosis (FHL)-5 patients, which have defective granule release due to a mutation in the

309 STXBP2 gene encoding the munc18-2 protein (37). Neutrophils from chronic granulomatous disease

310 (CGD) patients, which have mutations in components of the NADPH oxidase causing a complete lack

311 in the production of reactive oxygen species (38, 39). IgA-mediated trogocytosis and ADCC by

312 neutrophils of these patients was not abrogated, confirming that indeed neutrophil NADPH oxidase

313 and granules and their components do not form an absolute requirement for ADCC of IgA-opsonized

314 cancer cells (Fig. 3H-K).

315 To further evaluate the direct contribution of neutrophil-induced trogocytosis to FcαR-mediated

316 ADCC we used a number of selected inhibitors of intracellular targets (targeting the tyrosine kinase

317 Syk, PI3K, myosin light chain kinase, intracellular calcium) that we previously identified operating

318 downstream of FcR during IgG meditated neutrophil trogocytosis of cancer cells (40). Our results

319 demonstrated that these inhibitors, as well as CD11b/CD18 blocking antibodies, prevented IgA-

320 mediated trogocytosis and IgA-mediated ADCC in all of the tested cases (Fig. 4A, B). This was true

321 with or without CD47-SIRPα inhibition, indicating that inhibition of CD47-SIRPα directly promoted the

322 neutrophil intracellular signaling that drives these pathways of trogocytosis and/or killing, rather

323 than inducing alternative signaling downstream of the FcαR (Fig. 4A, B).

324

325 IgA induced cytotoxicity of tumor cells during inhibition of CD47-SIRPα interactions in mouse models

326 To further investigate the effect of blocking CD47-SIRPα interactions in combination with IgA

327 therapeutic antibody in an in vivo setting, we made use of mice expressing human FcαRI (9) on a SCID

328 background. With these mice, we established a long-term mouse xenograft model using the human

329 epidermoid cell line A431. We compared tumor growth of CD47 expressing A431 cells (A431 ctrl) to

330 A431-CD47KO. These two cell lines were injected in the right or left flank of the same mouse (Fig 5A).

331 Groups of mice were systemically treated with either PBS, cetuximab or anti-EGFR-IgA2 when

332 palpable tumors formed (day 6, Fig. 5A). After 17 days, tumor volume was only significantly reduced

333 in the CD47KO A431 tumors after anti-EGFR-IgA2 treatment compared to cetuximab, whereas

334 control tumors displayed no volume alteration (Fig. 5B, C).

335 To further examine the role of neutrophils as effector cells in vivo, we made use of a syngeneic

336 mouse model expressing human FcαRI (9) treated with the mouse SIRPα specific antagonistic

337 antibody MY-1 (Fig. S3A) (30). This antibody was used in our syngeneic in vivo model to only block

338 the interaction between CD47 and SIRPα without potentially causing opsonization of target cells, as

339 might occur with anti-CD47 antibodies (39,40). To determine the capacity of mouse neutrophils to

340 cause ADCC in the syngeneic tumor model , we first isolated mouse neutrophils from bone marrow of

341 FcαRI transgenic and wild type mice. The mouse pro-B cell line Ba/F3, which does not express mouse

342 SIRPα (Fig. S3B), was used as target, either transfected with human HER2/neu or EGFR (Fig. 6A).

343 Experimental conditions consisted of IgA2 or IgG1 antibodies against HER2/neu or EGFR in

344 combination with MY-1, blocking mouse SIRPα. No antibody-dependent neutrophil-mediated killing

345 of the target cell lines was observed in the presence of an intact CD47-SIRPα signaling axis (Fig. 6B,

346 C). However, the IgA2 variant of the anti-HER2/neu therapeutic antibody significantly enhanced

347 ADCC by neutrophils expressing FcαR (Fig. 6B, C). This effect could be further increased by SIRPα

348 checkpoint blockade for both HER2/neu and EGFR-expressing Ba/F3 cells (Fig. 6B, C). These data

349 indicated that the use of IgA therapeutic antibodies caused significantly higher ADCC compared to

350 IgG therapeutic antibodies, specifically after blocking SIRPα with MY-1, which enhanced killing of

351 HER2/neu and EGFR-expressing tumor cells by murine neutrophils.

352

353 MY-1 was used to block SIRPα in a syngeneic in vivo mouse model as previously described (9), where

354 we compared the efficacy of anti-HER2 IgG or IgA subclasses in the presence or absence of SIRPα

355 blockade. Fluorescent Ba/F3 and Ba/F3-HER2 cells were injected in the peritoneal cavity and

356 combined with a therapeutic antibody against HER2/neu in the presence or absence of SIRPα

357 inhibition. Saturation of the SIRPα receptor with MY-1 on both macrophages and neutrophils was

358 confirmed using flow cytometry (Fig. S3C, D). Comparable to the in vitro setting, blocking SIRPα in

359 vivo by MY-1 led to a significantly and substantially increased reduction in tumor load compared to

360 the use of either IgG or IgA therapeutic antibodies alone (Figure 6D). As previously reported (41), the

361 antibody-mediated reduction in tumor load was accompanied by a significant influx of granulocytes,

362 particularly in the condition where MY-1 was combined with therapeutic antibody against HER2/neu.

363 This neutrophil influx was more pronounced when using anti-HER2-IgA2 compared to trastuzumab

364 (Fig. 6E, F) (42). Only a small decrease in macrophages, no alterations in the influx of other leukocyte

365 populations, was detected under this condition (Fig. 6F and Fig. S3E-H). Together, our data showed

366 that checkpoint blockade of CD47-SIRPα increased the therapeutic potential of anti-HER2-IgA2 in a

367 syngeneic mouse model, which was accompanied by an increase in granulocyte recruitment to the

368 tumor site.

369 To confirm that neutrophils are the effector cell population responsible for the killing of the Ba/F3-

370 HER2 cells in our syngeneic mouse model, we depleted neutrophils by the use of anti-Ly6G antibody

371 (Fig. S4A). This depletion did not result in significant changes in other leukocyte populations (Fig. S4B-

372 E). When neutrophils were depleted from the mice, only limited ADCC occurred when using both

373 anti-EGFR-IgA2 and MY-1 (Fig. 6G). These results show that in this model neutrophils are the

374 dominant effector cells required for therapeutic clearance with anti-HER2-IgA2 in the presence of

375 CD47-SIRPα checkpoint inhibition.

376 Altogether, our results demonstrated that IgA-mediated tumor therapy was restricted by CD47-SIRPα

377 interactions in vitro and in vivo – using both syngeneic and xenogeneic mouse models. This

378 restriction could be therapeutically overcome by CD47 or SIRPα blocking antibodies.

379 Discussion

380 In this manuscript, we demonstrated that neutrophil-mediated cytotoxicity of cancer cells by IgA

381 antibodies against HER2/neu or EGFR was limited by CD47-SIRPα checkpoint inhibition, both in vitro

382 and in vivo. This new finding implies that CD47-SIRPα axis may be a promising strategy for enhancing

383 IgA-mediated anti-tumor effects with neutrophils as effector cells. For IgG, the combination of

384 therapeutic antibodies, including rituximab, cetuximab, and trastuzumab, and inhibition of CD47-

385 SIRPα interactions shows promising results in enhancing antibody therapy in several pre-clinical

386 mouse models (18, 20, 21, 43). Thus, CD47 antibodies and SIRPα-Fc proteins are currently being

387 tested in phase I/II clinical trials in combination with IgG therapeutic antibodies (24, 25). Our study

388 implies that the combination of innate checkpoint inhibition and IgA antibodies against tumor

389 antigens may be similarly promising.

390 We provide evidence that neutrophils are the dominant effector cells against IgA antibody-opsonized

391 cancer cells in our syngeneic in vivo model. This is in contrast to our previous work where

392 macrophages were the dominant effector cells eliminating IgA-opsonized tumor cells in Ba/F3 tumors

393 overexpressing EGFR (9). In this particular model the CD47-SIRPα axis was still intact, despite the use

394 of different systems and therapeutic antibodies. This could contribute to a different effector cell

395 population being activated and recruited against the antibody-opsonized cancer cells. Collectively,

396 our data demonstrate that, depending on the therapeutic antibody – antigen combination, both

397 neutrophils and macrophages can mediate cytotoxicity by IgA anti-tumor antibodies. These data

398 suggest that upon the additional blockade of the CD47-SIRPa axis, neutrophils are recruited and

399 contribute to the killing process. This neutrophil recruitment could potentially be the result of an IgA

400 induced feed-forward loop that depends on paracrine effects of neutrophil chemoattractants like

401 LTB4 (41).

402 Most research on the development of cancer immunotherapies has focused either on stimulating

403 antibody-mediated effector functions of the innate immune system or enhancing an adaptive

404 immune response. In the case of targeting CD47-SIRPα interactions, the initial focus of research has

405 been on activation of the innate immune system leading to increased ADCC by neutrophils and

406 enhanced ADCP by macrophages (18, 20, 21, 43). Checkpoint inhibition of the CD47-SIRPα axis

407 increases effective T-cell responses against tumors by activating CD8+ T cells and suppressing CD4+ T

408 cells (44). The mechanism by which CD47-SIRPα checkpoint inhibition stimulates a cytotoxic T cell

409 response against tumors could possibly involve enhanced macrophage- or myeloid dendritic cell-

410 mediated antigen presentation in response to an increased uptake of tumor material (44, 45), or

411 perhaps even an increase and contribution of neutrophil antigen presentation (46, 47). Although

412 further research is needed to confirm this possibility, combining IgA-based antibody therapy against

413 cancer with inhibition of CD47-SIRPα interactions could lead to an efficient long-lasting adaptive

414 immune response.

415 An important drawback of using IgA anti-cancer antibodies clinically is the relatively short

416 half-life of IgA antibodies in vivo (9), even though this drawback in half-life is more apparent in mice.

417 In mice, the neonatal Fc-receptor (FcRn) has a stronger binding capacity for human IgG, which

418 thereby exaggerates the half-life of human IgG compared to human IgA in mice(48). There may be

419 various ways to solve the relatively short half-life of IgA antibodies in humans. We and others have

420 described ways to extend the serum half-life of IgA antibodies via glyco-engineering strategies (49,

421 50) or by inducing their binding to the neonatal Fc-receptor (FcRn) via protein engineering (13, 51,

422 52). Another potential solution for increasing both the half-life and engaging an optimal population

423 of effector cells was suggested by Georgiou et al, who generated an antibody with an Fc-region that

424 could simultaneously bind both Fcγ-receptors, and FcαR, a so called IgGA-antibody (53). This

425 antibody engages NK cells, macrophages, monocytes, and neutrophils, and activates the complement

426 system (53). Future study is required to determine if combining engineered IgA antibodies with

427 inhibitors of CD47-SIRPα interactions are effective in triggering anti-cancer neutrophils.

428 Collectively, our findings show that IgA cancer immunotherapy is restricted by CD47-SIRPα

429 checkpoint inhibition, providing support for the idea that targeting the CD47-SIRPα checkpoint could

430 be used to potentiate the anti-tumor efficacy of IgA therapeutic antibodies in cancer.

431 Acknowledgements

432 We would like to thank dr. Robin van Bruggen for critically reading the manuscript and prof. dr.

433 Takashi Matozaki for providing the MY-1 hybridoma. Our research was supported by the following

434 grants: from Landsteiner Foundation for Blood Transfusion Research (LSBR-1223, awarded to T.K. van

435 den Berg), from the Dutch Cancer Society (#10300, awarded to T.K. van den Berg; #11537, awarded

436 to H.L. Matlung, and UU 2015-7650, awarded to J.H.W. Leusen), from Kika (#227, awarded to J.H.W.

437 Leusen) and from the German Research Organization (DFG Va124/9-1, awarded to T. Valerius). T. K.

438 van den Berg was supported by a research collaboration with Synthon Biopharmaceuticals related to

439 CD47-SIRPα targeting in cancer.

440 References

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582

583 Figure legends

584 Figure 1: Downregulation of CD47 expression promoted IgA mediated ADCC by neutrophils. (A)

585 Representative histograms (n=2) showing the expression of CD47 and HER2/neu, EGFR in SKBR3 and

586 A431 cells (upper panels) and their respective CD47KD or –KO variants (lower panels). (B) Genetic

587 disruption of CD47-SIRPα interactions led to increased ADCC of tumor cells (SKBR3, A431) by freshly

588 isolated neutrophils using increasing concentrations of the indicated IgG1 (black lines) or their IgA2

589 variants (grey lines). Data shown are means ± SEM pooled from 2-3 experiments with 2-3 donor

590 samples per experiment with n=2-5 (B, SKBR3), n=3-4 (B, A431), and n=3 (C) individual donors.

591 Statistics shown in (B) and (C) are for the highest antibody concentrations or highest E:T ratio using

592 two-way ANOVA with Tukey’s correction for multiple tests. ***p < 0.001.

593 Figure 2: Different approaches to inhibit CD47-SIRPα interactions enhanced IgA mediated ADCC.

594 ADCC of cancer cells by freshly isolated neutrophils without (white background) and with inhibition

595 of CD47-SIRPα interactions (grey background) by either using cell lines with no (A431-CD47KO) or

596 reduced (SKBR3-CD47KD) expression of CD47 (A), inhibition of CD47 with B6H12 F(ab‘)2 antibodies

597 (B) or inhibition of SIRPα using antibody 12C4 (C). Used are either anti-HER2-IgA2 antibody against

598 SKBR3 or anti-EGFR-IgA2 antibody (both at 5 µg/mL) against A431 cells, respectively. Data shown are

599 means ± SEM pooled from 2-3 experiments with 2-3 individual donor samples per experiment with

600 n=8 (B), n=7-8 (C), n=6-8 (D). Statistics were performed by paired t-test. **p < 0.01.

601

602 Figure 3: IgA-mediated trogocytosis of cancer cells by neutrophils. Representative live cell imaging

603 stills (A-F) at different time points of a neutrophil binding to an anti-HER2-IgA2 opsonized cancer cell

604 and trogocytosing pieces of fluorescent cancer cell membrane. Scale bar = 10 μm. Trogocytosis of

605 SKBR3 cells (G) opsonized with increasing concentrations of trastuzumab or anti-HER2-IgA2. (H) ADCC

606 and (I) trogocytosis of anti-EGFR-IgA2 opsonized A431 cancer cells by freshly isolated neutrophils

607 isolated from healthy controls or FHL5 patients (for patient characteristics see patients B and C

608 described in (32)) combined without (white background) or with CD47-SIRPα inhibition by knockout

609 of CD47 in the target cells (grey background). (J) ADCC and (K) trogocytosis of anti-HER2-IgA2

610 opsonized SKBR3 cells by freshly isolated neutrophils isolated from healthy controls or CGD patients

611 (for patient characteristics see methods), combined without (white background) or with CD47-SIRPα

612 inhibition by knockdown of CD47 in the target cells (grey background). Data shown are means ± SEM

613 pooled from 2 separate experiments with 2 replicates of 2 individual FHL5 patient samples and 11

614 total healthy controls (H)-(I), 3 separate experiments with 3 (K) or 4 (J) individual CGD patient

615 samples and 4 day controls (J)-(K), or pooled from 2 experiments with 1-2 donor samples per

616 experiment with n=4 (G) individual donors. Statistics shown in (G) are for the highest antibody

617 concentrations using two-way ANOVA with Tukey’s correction for multiple tests. No statistics could

618 be performed on FHL5 patient data in (H)-(I) due to low patient count (n=2). Statistics shown in (J)-(K)

619 using one-way ANOVA, with Sidak correction for multiple tests. ns = non-significant, *p < 0.05, ***p <

620 0.001.

621

622 Figure 4: Inhibition of CD47-SIRPα did not result in alternative FcαR-mediated ADCC

623 Neutrophil mediated trogocytosis (A) and ADCC (B) of SKBR3 and SKBR3-CD47KD cells using IgA2-

624 Her2 antibody with inhibition of Syk, PI3K, myosin light chain kinase, CD11b/CD18- integrins and

625 calcium mobilization. Data shown are means ± SEM pooled from 2 experiments with 2 donor samples

626 per experiment with n=4 individual donors. Statistics shown using one-way ANOVA, with Sidak

627 correction for multiple tests. *p<0.05, ****p<0.001.

628

629 Figure 5: Interference with CD47-SIRPα interactions enhanced tumor eradication in a xenogeneic

630 long-term in vivo model. (A) Schematic overview of the in vivo xenograft model and the injection

631 scheme. (B-C) Long-term in vivo tumor growth comparing the maternal A431-SCR cell line (B) to one

632 with no expression of CD47 (A431-CD47KO) (C). Treatment was started when mice had palpable

633 tumors (day 6) with either PBS (light grey line), a single iv injection of 50 µg cetuximab (black line), or

634 an iv injection of 250 µg anti-EGFR-IgA2 followed by 4 i.p. injections of 250 µg (dark grey line) to

635 compensate for the shorter half-life of IgA compared to IgG. Tumor outgrowth was measured with

636 calipers and volume was calculated as length x width x height. Data shown are means ± SEM from 1

637 experiment with 8 mice per group in (B) and (C). Statistics performed are for day 17 using two-way

638 ANOVA with Tukey’s correction for multiple tests. ns = non-significant, *p < 0.05.

639 Figure 6: Inhibition of SIRPα effectively triggered syngeneic tumor cell killing by neutrophils. (A)

640 Expression of CD47 and HER2 or EGFR in Ba/F3 cells transfected with human HER2 or EGFR,

641 respectively. (B) ADCC of trastuzumab or anti-HER2-IgA2 (both at 5 µg/mL) opsonized Ba/F3-HER2

642 cells by mouse neutrophils isolated from wildtype (NTg) or FcαR-transgenic (FcαR-Tg) mice combined

643 without (white background) or with inhibition of SIRPα (MY-1) (grey background). (C) Same as (B),

644 but with cetuximab or anti-EGFR-IgA2 opsonized Ba/F3-EGFR cells. (D) In vivo inhibition of SIRPα (MY-

645 1) was combined with trastuzumab or anti-HER2-IgA2 treatment in a short intraperitoneal model.

646 The ratio of Ba/F3-HER2 and Ba/F3 cells in wildtype mice (PBS, Isotype+Tmab, anti-SIRPα (MY-

647 1)+Tmab) or FcαR-transgenic mice (Isotype+anti-HER2-IgA2, anti-SIRPα (MY-1)+anti-HER2-IgA2) in

648 the peritoneal washes was determined by flow cytometry. (E) Number of granulocytes

649 (Ly6G+/CD11b+) present in the peritoneal cavity at the end of the experiment in (D). (F) Overview of

650 all cells present in the peritoneal cavity during the experiment shown in (D). (G) Inhibition of SIRPα

651 (MY-1) is combined with anti-HER2-IgA2 and neutrophil depletion (Ly6G) in an in vivo mouse

652 experiment, showing the ratio of Ba/F3-HER2 and Ba/F3 cells in wildtype mice (Isotype, anti-SIRPα

653 (MY-1), anti-SIRPα(MY-1) +anti-HER2-IgA2 (NTg)) or FcαR-transgenic mice (Isotype+anti-HER2-IgA2,

654 anti-SIRPα (MY-1)+anti-HER2-IgA2, anti-SIRPα (MY-1)+anti-HER2-IgA2+Ly6G). Data shown for (B-C)

655 are means ± SEM pooled from 2 experiments with 2 mice per experiment with a total of n=4

656 individual mice. Data shown for (D-F) are means ± SEM from 1 experiment with 6 mice per group

657 with (A) representative of n=2, (B) n=3-6, (C) n=6, (D) n=6 individual mice. Statistics shown for (B-C)

658 are calculated by one-way ANOVA, with Dunnett’s correction for multiple tests. Statistics shown for

659 (D) and (G) are calculated by paired one-way ANOVA with Sidak’s correction for multiple tests.

660 Statistics performed for (E) are calculated by one-way ANOVA with Sidak’s correction for multiple

661 tests. ns= non-significant, *p < 0.05, and **p < 0.01, ***p < 0.001.

IgA mediated killing of tumor cells by neutrophils is enhanced by CD47-SIRP α checkpoint inhibition

Louise W. Treffers, Toine Ten Broeke, Thies Rösner, et al.

Cancer Immunol Res Published OnlineFirst November 5, 2019.

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