Immune receptor inhibition through enforced phosphatase recruitment

ABSTRACT Antibodies that antagonize extracellular receptor–ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However,

this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for

attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels _cis_-ligation of cell-surface receptors containing ITAM,

ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the

receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by

ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling.

RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour

models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the

macrophage SIRPα ‘don’t eat me’ signal with a SIRPα–CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general

strategy for direct attenuation of signalling by kinase-activated cell-surface receptors. Access through your institution Buy or subscribe This is a preview of subscription content, access

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DEVELOPMENT OF A 1:1-BINDING BIPARATOPIC ANTI-TNFR2 ANTAGONIST BY REDUCING SIGNALING ACTIVITY THROUGH EPITOPE SELECTION Article Open access 27 September 2023 PD-1 SUPPRESSES TCR-CD8

COOPERATIVITY DURING T-CELL ANTIGEN RECOGNITION Article Open access 12 May 2021 DATA AVAILABILITY Data that support the findings of this study are available from the corresponding author

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alpha and methods of use. WIPO patent WO2018057669A1 (2016). Download references ACKNOWLEDGEMENTS We thank members of the Garcia Laboratory for advice and discussion, and M. M. Davis for

support with the MC38 tumour model. This work was supported by the Howard Hughes Medical Institute and National Institute (K.C.G.), NIH (R01 AI103867 to K.C.G., U01 CA213273 and R35

CA2311997 to J.S., K00 CA223019 to N.C.), Mathers Foundation (K.C.G.), Stanford Cancer Institute seed grant (K.C.G.), Ludwig Foundation (K.C.G.), Wellcome Trust (Sir Henry Wellcome

Fellowship, WT101609MA to R.A.F.), Taube Distinguished Scholar for Pediatric Immunotherapy at Stanford University School of Medicine (R.G.M.), Mentored Clinical Scientist Development Award

1K08DK114563-01 (M.D.) and the American Gastroenterological Association Research Scholars Award (M.D.) AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular and Cellular

Physiology, Stanford University School of Medicine, Stanford, CA, USA Ricardo A. Fernandes, Leon Su, Junming Ren, Lora K. Picton, Shozo Ohtsuki, Xinbo Yang, Christina S. Savvides & K.

Christopher Garcia * Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA Ricardo A. Fernandes, Leon Su, Junming Ren, Lora K. Picton, Shozo Ohtsuki, 

Xinbo Yang, Christina S. Savvides & K. Christopher Garcia * Department of Pediatrics, Stanford University, Stanford, CA, USA Yoko Nishiga, Robbie G. Majzner, Crystal L. Mackall & 

Julien Sage * Department of Radiation Oncology, Stanford University, Stanford, CA, USA Yoko Nishiga * Department of Medicine, Division of Gastroenterology, Massachusetts General Hospital,

Boston, MA, USA Aladdin M. Bhuiyan & Michael Dougan * Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA Ning Cheng & Calvin J.

Kuo * Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA Robbie G. Majzner, Skyler P. Rietberg & Crystal L. Mackall * Department of Medicine, Stanford

University School of Medicine, Stanford, CA, USA Crystal L. Mackall * Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA Qian

Yin * Department of Medicine, Harvard Medical School, Boston, MA, USA Lestat R. Ali & Michael Dougan * Department of Genetics, Stanford University, Stanford, CA, USA Julien Sage * Howard

Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA K. Christopher Garcia Authors * Ricardo A. Fernandes View author publications You can also search for

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You can also search for this author inPubMed Google Scholar * K. Christopher Garcia View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.A.F.

and K.C.G. conceived the project and wrote the manuscript. K.C.G. supervised the research. Y.N. performed and analysed the experiments with SCLC tumour cells with help from R.A.F. and L.S.

under the guidance of J.S. R.A.F. and L.S. performed T cell experiments in vitro and in vivo and prepared reagents for in vivo studies. R.A.F. and L.S. performed experiments with MC38 tumour

cells, with guidance from Q.Y. J.R. performed phagocytosis experiments. A.M.B. and L.R.A. performed in vitro experiments under the guidance of M.D. R.G.M. and S.P.R. performed CAR T

experiments under the guidance of C.L.M. R.A.F. and S.O. performed signalling reconstitution experiments in HEK293 cells. N.C. performed organoid studies under the supervision of C.J.K.

R.A.F and X.Y. performed surface plasmon resonance measurements. R.A.F., C.S.S. and L.K.P. expressed proteins for in vivo and in vitro studies. CORRESPONDING AUTHOR Correspondence to K.

Christopher Garcia. ETHICS DECLARATIONS COMPETING INTERESTS RIPR is the subject of a pending patent application (63/056,156), with K.C.G. and R.A.F. listed as inventors. K.C.G. is the

founder of Synthekine Therapeutics Inc. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature_ thanks Enfu Hui and the other, anonymous, reviewer(s) for their contribution to the peer

review of this work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA FIGURES AND TABLES

EXTENDED DATA FIG. 1 TONIC PD-1 SIGNALLING DAMPENS T CELL ACTIVATION. A, TCR downregulation in wild-type or transduced Jurkat T cells with CRISPR/Cas9 and gRNA targeting PD-1 (J-CRISPR-PD-1)

upon OKT3 stimulation overnight. B, Jurkat T cells transduced with wild-type human PD-1 (Jurkat-PD1). C, PD-1 versus CD69 expression for Jurkat T cells stimulated overnight with OKT3. Data

are shown as individual biological replicates, _n_ = 3. D, CD69 upregulation after OKT3 and anti-CD28 stimulation overnight of wild-type Jurkat cells or Jurkat-PD1 cells (shown in B) or PD-1

KO cells (J-CRISPR-PD-1). In A, D, data are shown as mean ± s.d., _n_ = 3 biological replicates from 1 representative of 3 independent experiments. E, F, Jurkat cells were transduced with

control gRNA/CRISPR plasmid or gRNA targeting the endogenous PD-1 gene and activated at increasing concentrations of plate-bound OKT3 overnight. In a separate control group, Jurkat cells

were treated with anti-PD1 (Nivolumab), anti-PD-L1 and anti-PD-L2 antibodies (50 μg ml−1 for each antibody). CD69 (E) and PD-1 (F) expression was quantified by flow cytometry. Data are mean 

± s.d. from _n_ = 2 biological replicates from 1 representative of 2 independent experiments. G, Expression of HA–PD1-ICD (ECD-less) reduced the upregulation of CD69 12 h after activation

with plate-bound OKT3 at the indicated concentrations. Data are mean ± s.d. from _n_ = 3 biological replicates from 1 representative of 2 independent experiments. H, I, Quantification of

PD-L1 and PD-L2 in resting and activated Jurkat cells. Resting or activated Jurkat T cells, were incubated with anti-PD-PL1 (H), anti-PD-L2 (I) or with appropriate isotype controls and

analysed by flow cytometry. Expression of PD-L1 and PD-L2 was not detected in either resting or activated Jurkat T cells. Non-fluorescent anti-PD-L1 or L2 antibody (right) was added before

labelling as a ‘blocking’ control. In H, I, data are representative of 3 independent experiments. Source data EXTENDED DATA FIG. 2 DEVELOPMENT AND IN VITRO TESTING OF RIPR-PD1. A,

Coomassie-stained SDS–PAGE of SEC-purified RIPR-PD1(nivo). B, C, Curve fitting of recorded resonance units at steady state for RIPR-PD1 binding to immobilized CD45 (B) and PD-1 (C). Data

shown for RIPR-PD1 concentrations ranging from 4 μM to 35 nM (B), and from 8 μM to 17.5 nM (C). Data are representative of 2 independent experiments. D, E, Fraction of CD25+CD69+ (D) and

IL-2 (E) expression for wild-type Jurkat T cells stimulated with 2 μg ml−1 of plate-bound OKT3 and treated with nivolumab or RIPR-PD1 at the concentrations indicated for 24 h. F–I,

Quantification of CD25 (F) or CD69 (G) expression and fraction of CD25+CD69+ (H) for Jurkat T cells transduced with wild-type PD-1 (shown in Extended Data Fig. 1b) and IL-2 secretion (I) for

SKW-3 T cells stimulated with 2 μg ml−1 of plate-bound OKT3 and treated with nivolumab or RIPR-PD1 at the indicated concentrations for 24 h. J, Quantification of IL-2 secretion by TCR

transduced SKW-T cells after stimulation for 24 h with cognate peptide in the presence or absence of PD-L1 and nivolumab or RIPR-PD1 at the indicated concentrations. K, L, IFNγ (K) and IL-2

(L) secretion by indicated CAR T cells (or mock untransduced T cells) after incubation with tumour target cells in the presence of increasing concentrations of RIPR-PD1. In D–L, data are

mean ± s.d. from _n_ = 3 biological replicates representative of 3 independent experiments. Source data EXTENDED DATA FIG. 3 ANTIGEN STIMULATION OF PBMCS IS POTENTIATED BY RIPR MOLECULES. A,

B, Peptide-pulsed PBMCs (PepMix, JPT) were treated with 1 μM of nivolumab, pembrolizumab, RIPR-PD1 with nivo (N) or pembro (P) scFv and anti-CD45 diabody (anti-CD45-Db#4). CD69 was analysed

by flow cytometry and IFNγ was quantified by ELISA. Data are mean ± s.d. from _n_ = 3 biological replicates from 1 representative of 2 independent experiments. C, CFSE-labelled CD8+ human T

cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of nivolumab or RIPR-PD1 at 1 μM. T cell proliferation was analysed by FACS on day 3. Data are mean ± s.d. from

_n_ = 4 (untreated and anti-CD3/CD28) or _n_ = 9 (nivolumab and RIPR-PD1) biological replicates from 1 representative of 2 independent experiments. Source data EXTENDED DATA FIG. 4 RIPR-PD1

BINDS TO CD45 AND PD-1 ECTODOMAINS. A, B, Size-exclusion chromatography profile (A) and Coomassie-stained SDS–PAGE (B) of RIPR-PD1(VHH). C–F, SPR binding curves for RIPR-PD1(VHH) binding to

CD45 (C) and PD-1 (E), and curve-fitting to resonance units measured at steady state for multiple RIPR-PD1 concentrations during binding to immobilized CD45 (D) and PD-1 (F). In C, D, data

shown is for RIPR-PD1 concentrations ranging from 9 μM to 35 nM. In E, F, data shown is for RIPR-PD1 concentrations ranging from 250 nM to 3.5 nM. _K_d values were found to be around 700 nM

for binding to CD45 and around 3 nM for binding to PD-1. Data shown are representative of 2 independent experiments. G, Jurkat T cells were stimulated with OKT3 at the indicated

concentrations and treated with 1 μM of nivolumab, RIPR-PD1(nivo), RIPR-PD1(VHH) or anti-CD45 diabody (anti-CD45-Db#4). CD69 (left) and CD25 (right) were quantified by FACS 24 h after

stimulation. Data are mean from _n_ = 2 biological replicates representative of 3 independent experiments. H, SPR analysis of RIPR-PD1 simultaneously binding to CD45 and PD-1. Biotinylated

human PD-1 was immobilized in two independent flow cells using a streptavidin coated surface (top). RIPR-PD1(VHH) was used as an analyte at 1 μM in channel 2 (Fc2; middle). CD45 was then

used as an analyte in both channels at the indicated concentrations (bottom). Binding of CD45 was detected only in the channel in which both PD-1 and RIPR were present, Fc2, indicating that

RIPR-PD1(VHH) bound to PD-1 was able to bind CD45. Fc, Flow cell. Data are representative of 2 independent experiments. Source data EXTENDED DATA FIG. 5 RIPR-PD1 POTENTIATES T CELL EXPANSION

IN TUMOUR ORGANOIDS, AND RIPR ACTIVITY IS NOT STRICTLY DEPENDENT ON PD-1 BLOCKADE. A, Quantification of TILs treated with intact RIPR (hRIPR-PD1(VHH)), 3C-digested control RIPR-PD1(VHH)

(black) or nivolumab (nivo) in melanoma (left) or ovarian (right) patient-derived tumour organoids, at the indicated concentrations. B, Activated Jurkat T cells upregulate PD-1 (_x_ axis)

and CD69 (_y_ axis). Anti-PD-1 staining is blocked by RIPR-PD1(VHH) (left) but is not affected by non-blocking RIPR-PD1(Cl19) (right). C, HEK293 cells were transfected with HA–PD-1 (N-term

HA-tag fused to full-length PD-1) and LCK and CD45, as indicated. 48 h after transfection cells were treated with RIPR-PD1(VHH) or RIPR-PD1(Cl19) for 30 min at 37 °C at 0.5 or 1 μM, or

3C-cleaved RIPR-PD1(VHH) (1 μM) as indicated. Anti-HA magnetic beads were used for immunoprecipitation and samples were probed for anti-phosphotyrosine or anti-PD1 by western blot. Data are

representative of two independent replicates. For raw source image, see Supplementary Fig. 1. D, E, Quantification of CD69 expression (D) and IFNγ secretion (E) after PBMC stimulation (as

described in Fig. 3) for cells treated with 1 μM of nivolumab, RIPR-PD1(nivo) or non-blocking RIPR-PD1(Cl19). Data are mean ± s.d. from _n_ = 3 biological replicates from 1 representative of

2 independent experiments. For representative gating strategy, see Supplementary Fig. 2. F, Jurkat T cells were activated overnight with plate-bound OKT3 at 0.1 μg ml−1 and incubated with 1

μM of RIPR-PD1(VHH) (blocking) or RIPR-PD1(Cl19) (non-blocking). Data are mean from _n_ = 2 biological replicates from 1 representative of 2 independent experiments. G, Size exclusion

chromatography of mouse RIPR-PD1. H, SPR binding curves for mRIPR-PD1 (clone F2) binding to immobilized mouse PD-1. I, Resonance units measured at steady state for multiple mRIPR-PD1

concentrations tested for binding to mouse PD-1. RIPR-PD1 concentration shown ranges from 8 μM to 35 nM. _K_d values were found to be around 750 nM for binding to mPD-1. In H, I, data are

representative from 2 independent experiments. J, Mouse CD8+ T cells were isolated from spleen and lymph nodes of C57/B6 mice and stimulated with plate-bound anti-CD3 (2C11) antibody at the

indicated concentrations plus soluble CD28 (2 mg ml−1). During activation with 2C11 and anti-CD28, cells were either left untreated or were incubated with anti-PD-1 antibody (clone RMP1-14)

or mouse RIPR-PD1(RMP1-14) for 24 h. CD69 (top) and CD25 (bottom) expression was quantified by FACS. K, Pmel-1 mouse CD8 T cells were stimulated with peptide pulsed APCs and 250 nM of

anti-PD1 (RMP1-14) antibody or RIPR-PD1(RMP) for 24 h. CD69 were analysed by flow cytometry. In J, K, data are mean ± s.d. from _n_ = 2 biological replicates from 1 representative of 3

independent experiments. Source data EXTENDED DATA FIG. 6 IN VITRO TESTING OF MOUSE RIPR-PD1. A–E, CD4+ T cells were stimulated for 3 days in the presence of anti-CD3/CD28 and anti-PD1,

anti-PD-L1(VHH) or RIPR-PD1, all at 1 μM, after which cells and supernatant were collected by analysis for proliferation (A), IL-2 (B) or IFNγ (C) secretion, fraction of CD44highCD62Llow (D)

and CD25 (E). Cell proliferation was quantified using CellTiter-Glo, surface expression by flow cytometry and cytokine secretion by ELISA. F, Mouse CD4+ T cells were stimulated with

plate-bound anti-CD3 and soluble CD28 in the presence of absence of plate-bound PD-L1 or isotype control. Cells were treated with anti-PD1 antibody (RMP1-14) or RIPR-PD1 at 1 μM and

supernatant was collected for quantification of IFNγ production on day 3. In A–F, data are mean ± s.d. from _n_ = 3 biological replicates from 1 representative of 2 independent experiments.

G, Size-exclusion chromatography of intact or 3C-cleaved mouse RIPR-PD1. H, Coomassie-stained SDS–PAGE of mouse mRIPR-PD1 uncleaved or treated with 3C overnight. I, Mouse CD8+ T cells were

activated with plate-bound 2C11 (1 mg ml−1) and soluble CD28 (2 mg ml−1) for 24 h and treated with 1 μM of intact (middle) or 3C-treated (right) mRIPR-PD1. J, K, Quantification of the CD69

(J) and CD25 (K) expression of representative data shown in I. Data are mean from _n_ = 2 biological replicates from 1 representative of 3 independent experiments. L, Competition experiment

between mRIPR-PD1 at the indicated concentrations and anti-CD45-MSA (2 μM), anti-PD-1-MSA (2 μM) or both, as indicated. Mouse CD8+ T cells were stimulated with 8 μg ml−1 of 2C11 and 2 μg

ml−1 of anti-CD28. Black line indicates CD69 expression (with 2C11 and anti-CD28 only). CD69 expression was quantified by FACS 24 h post-stimulation. Data are mean from _n_ = 2 biological

replicates from 1 representative of 3 independent experiments. M, N, Competition experiment between mRIPR-PD1 and soluble mouse PD-1 (5 μM). Mouse CD8+ T cells were stimulated with 2 μg ml−1

(M) or 4 μg ml−1 (N) of plate-bound 2C11 and 2 μg ml−1 of soluble anti-CD28. CD69 was quantified by FACS 24 h after stimulation. Data are mean from _n_ = 2 biological replicates from 1

representative of 3 independent experiments. Source data EXTENDED DATA FIG. 7 RIPR-PD1 INDUCES T CELLS WITH AN EFFECTOR MEMORY PHENOTYPE IN A SCLC TUMOUR MODEL. A, Representative analysis of

CD44 and CD62L expression in lymphocytes isolated from tumour-proximal lymph nodes collected at day 19 post inoculation with KP1 (SCLC) cells for various treatments, as indicated. Data are

representative from _n_ = 5 biological replicates from 2 independent experiments. B, C, Quantification of the representative data shown in A for CD4+ (B) and CD8+ (C) cells. In B, C, data

are mean ± s.d. from _n_ = 5 biological replicates representative of 2 independent experiments. D, Quantification of the frequency of CD4+ and CD8+ cells in spleen (left) peripheral (middle)

and distal (right) lymph nodes from wild-type mice treated with 200 μg anti-PD1 or RIPR-PD1 every 3 days for one week. E, Quantification of CD4+ and CD8+ infiltrates in brown adipose

tissue. Mice were treated as described in D. In D, E, data are mean ± s.d. from _n_ = 3 (D) and _n_ = 5 (E) biological replicates representative of 2 independent experiments. For

representative gating strategy, see Supplementary Fig. 2. Source data EXTENDED DATA FIG. 8 RIPR-PD1 REDUCES MC38 COLON CARCINOMA TUMOUR GROWTH. A, B, Analysis of the RIPR-PD1(F2) binding

affinity to mouse PD-1 by SPR. C, D, Analysis of RIPR-PD1(RMP) binding affinity to mouse PD-1 by SPR. In A–D, data are representative of 2 independent experiments. CD8+ mouse T cells were

stimulated with plate-bound 2C11 overnight. E, F, Quantification of CD69 upregulation for cells treated with RIPR-PD1(F2) (E) or RIPR-PD1(RMP) (F). G, Proliferation (CellTiter-Glo) analysis

of anti-CD3 treated CD4+ mouse T cells in the presence of F2 or RMP RIPR-PD1. In E–G, data are mean ± s.d. from _n_ = 2 biological replicates from 1 representative of 3 independent

experiments. H, MC38 tumour growth for mice treated with PBS, anti-PD1 (clone RMP1-14, 200 μg every 3 days) or RIPR-PD1(RMP) (200 μg daily). Data are mean ± s.e.m. from _n_ = 10 mice. I,

Representative images of the mice 24 days after inoculation with MC38 cells for the indicated treatments. Data shown are representative of 2 independent experiments, _n_ = 10 mice per group.

J, Individual tumour volume measurements for the data shown in Fig. 4f. The _n_ values stated are the number of tumour-free mice. K, Percent survival for mice treated as described in H–J.

In H–J, data shown are representative of 2 independent experiments, _n_ = 10 mice per group. Source data EXTENDED DATA FIG. 9 PROFILING OF MOUSE T CELLS IN RESPONSE TO ANTI-PD1 OR RIPR-PD1

TREATMENT. A, Mice inoculated with MC38 tumour cells were treated with PBS, anti-PD1 or RIPR-PD1(RMP), 200 μg, every 3 days starting at day 5 post tumour inoculation. On day 12 T cells from

spleen (Spl), peripheral and distal lymph nodes (pLN, dLN) and tumour infiltrating lymphocytes (TILs) were collected and analysed by flow cytometry. B, Quantification of tumour weight. C,

Quantification of PD-1+ CD4+ T cells in TILs. D–F, Quantification of the fraction of positive CTLA-4 (D), TIM-3 (E) and LAG3 (F) from TILs for CD4+ T cells. G, H, Quantification of the

fraction of CD62LlowCD44high effector memory CD8+ T cells isolated from the spleen (G) or tumour (H). I, Quantification of the fraction of CXCR3-positive cells for splenic CD8+ cells.

Analysis of CD4+ cells showed increased fraction of CD137 (4-1BB) (J) and CXCR3 (K) in TILs. L, Quantification of effector memory and PD-1+ cells in tumour-free mice. Mice were treated with

PBS, anti-PD1 or RIPR-PD1 every 3 days from day 0 to day 6 and T cells were isolated and analysed on day 7. M–R, CD8+ and CD4+ T cells were analysed on day 7 for CD44, CD62L (M, N; CD8+,

spleen) and PD-1 expression in the spleen (O, P) and lymph nodes (mLN; Q, R). S, Quantification of Treg cells in tumour-free mice. Tumour-free FoxP3–GFP mice were treated with PBS, anti-PD1

or RIPR-PD1 (200 μg every 3 days) from day 0 to day 6, _n_ = 3 mice per group. T cells were analysed on day 7. Quantification of the fraction of FoxP3–GFP-positive cells (gated on the

CD3+CD4+ population) for cells isolated from the spleen. All data are mean ± s.d. from _n_ = 5 mice (B–K, M–Q) or _n_ = 3 (S) representative from 2 independent experiments. For

representative gating strategy, see Supplementary Fig. 2. Source data EXTENDED DATA FIG. 10 RIPR-SIRPΑ DEVELOPMENT AND IN VITRO TESTING. A, B, HEK293 cells were transiently transfected with

target receptors and LCK (A), or LCK plus CD45 (B). After lysis, chimeric receptors were immunoprecipitated with anti-HA antibody directly conjugated to magnetic beads. Samples were probed

for phosphotyrosine and HA by western blot. Data are representative of three independent biological repeats. For raw source image, see Supplementary Fig. 1. C, RIPR-SIRPα(3C) was treated

with 3C (100 μg ml−1) for 14 h at 4 °C. 3C-digestion was analysed by Coomassie blue staining on SDS–PAGE gel. D, Human PBMC macrophages were pretreated with RIPR-SIRPα or ‘Velcro’ protein

for 30 min at 37 °C and incubated with human tumour cells (Raji) pretreated with varying concentrations of rituximab (from 0 to 5 μg ml−1) for 30 min at 37 °C. Macrophages were co-cultured

with 1 × 104 CFSE+ Raji cells for 2 h at 37 °C. E, Quantification of phagocytosis as described in D for a fixed concentration of rituximab (1 μg ml−1) for cells treated with 100 nM or 500 nM

of intact or 3C-digested RIPR-SIRPα or Velcro, as indicated. In D, E, data are mean ± s.d. from _n_ = 2 (D) or _n_ = 3 (E) biological replicates representative from 2 independent

experiments. For representative gating strategy, see Supplementary Fig. 2. F, Coomassie blue staining on SDS–PAGE gel for AB21 Fab and RIPR-SIRPα(AB21). G, HEK293 cells were transiently

transfected with LCK, CD45, CD45dead and SIRPα as indicated. HEK293 cells were treated with RIPR-SIPRα 24 h after transfection for 30 min at 37 °C. Cells were collected and cell lysates were

incubated with HA beads, HA–SIRPα was immunoprecipitated and analysed by western blot assay as indicated. Data are representative from 2 independent experiments. For raw source image, see

Supplementary Fig. 1. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Raw uncropped western blot source images for data shown in the main and Extended Data Figures are shown in

Supplementary Figure 1. Boxes indicate cropping for representative images. Representative gating strategies used to analyze primary cells from mouse and human origin and Jurkat cells are

shown in Supplementary Figure 2. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 SOURCE DATA FIG. 2 SOURCE DATA FIG. 3 SOURCE DATA FIG. 4 SOURCE DATA FIG. 5 SOURCE DATA EXTENDED DATA FIG. 1

SOURCE DATA EXTENDED DATA FIG. 2 SOURCE DATA EXTENDED DATA FIG. 3 SOURCE DATA EXTENDED DATA FIG. 4 SOURCE DATA EXTENDED DATA FIG. 5 SOURCE DATA EXTENDED DATA FIG. 6 SOURCE DATA EXTENDED

DATA FIG. 7 SOURCE DATA EXTENDED DATA FIG. 8 SOURCE DATA EXTENDED DATA FIG. 9 SOURCE DATA EXTENDED DATA FIG. 10 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Fernandes, R.A., Su, L., Nishiga, Y. _et al._ Immune receptor inhibition through enforced phosphatase recruitment. _Nature_ 586, 779–784 (2020).

https://doi.org/10.1038/s41586-020-2851-2 Download citation * Received: 21 June 2019 * Accepted: 24 July 2020 * Published: 21 October 2020 * Issue Date: 29 October 2020 * DOI:

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