Chimeric antigen receptors that trigger phagocytosis
Chimeric antigen receptors ( CARs ) are synthetic transmembrane receptors that redirect T cell activity towards clinically relevant targets ( reviewed in [Lim et al . , 2017; Fesnak et al . , 2016] ) . The CAR-T receptor contains an extracellular single chain antibody fragment ( scFv ) that recognizes known tumor antigens , and intracellular signaling domains from the T Cell Receptor ( TCR ) and costimulatory molecules that trigger T cell activation ( Fesnak et al . , 2016; Kochenderfer et al . , 2009 ) . CAR-T cells recognizing CD19 , a marker expressed at high levels on the surface of B cells and B cell-derived malignancies , have been used successfully to target hematological malignancies with 70–90% of patients showing measurable improvement ( Lim et al . , 2017; Engel et al . , 1995; Haso et al . , 2013 ) . The success of CAR-T suggests that programming immune cells to target cancer might be a broadly applicable approach . Macrophages are critical effectors of the innate immune system , responsible for engulfing debris and pathogens . Harnessing macrophages to combat tumor growth is of longstanding interest ( Alvey and Discher , 2017; Lee et al . , 2016 ) . Macrophages are uniquely capable of penetrating solid tumors , while other immune cells , like T cells , are physically excluded or inactivated ( Lim et al . , 2017; Lee et al . , 2016 ) . This suggests that engineered macrophages may augment existing T cell-based therapies . Early efforts transferring healthy macrophages into cancer patients failed to inhibit tumor growth , suggesting that macrophages require additional signals to direct their activity towards tumors ( Lacerna et al . , 1988; Andreesen et al . , 1990 ) . Antibody blockade of CD47 , a negative regulator of phagocytosis , reduced tumor burden , indicating that shifting the balance in favor of macrophage activation and engulfment is a promising therapeutic avenue ( Majeti et al . , 2009; Chao et al . , 2010; Jaiswal et al . , 2009; Tseng et al . , 2013 ) . Here , we report a family of chimeric antigen receptors that activate phagocytosis of cancer cells based on recognition of defined cell surface markers , resulting in significantly reduced cancer cell growth .
To program engulfment towards a target antigen , we created a CAR strategy using the CAR-T design as a guide ( Fesnak et al . , 2016 ) . We call this new class of synthetic receptors Chimeric Antigen Receptors for Phagocytosis ( CAR-Ps ) . The CAR-P molecules contain the extracellular single-chain antibody variable fragment ( scFv ) recognizing the B cell antigen CD19 ( ɑCD19 ) and the CD8 transmembrane domain present in the ɑCD19 CAR-T ( Fesnak et al . , 2016; Kochenderfer et al . , 2009 ) . To identify cytoplasmic domains capable of promoting phagocytosis , we screened a library of known murine phagocytic receptors: Megf10 ( Figure 1a ) , the common ɣ subunit of Fc receptors ( FcRɣ ) , Bai1 , and MerTK ( Penberthy and Ravichandran , 2016 ) . FcR triggers engulfment of antibody-bound particles , while the other receptors recognize apoptotic corpses ( Freeman and Grinstein , 2014; Penberthy and Ravichandran , 2016 ) . We also made a receptor containing an extracellular ɑCD19 antibody fragment and a cytoplasmic GFP , but no signaling domain , to test whether adhesion mediated by the ɑCD19 antibody fragment is sufficient to induce engulfment ( Figure 1a; CAR-PGFP ) . To assay our library of CAR-Ps , we introduced each CAR-P into J774A . 1 murine macrophages by lentiviral infection . As an engulfment target , we used 5 µm diameter silica beads coated with a supported lipid bilayer . A His8-tagged extracellular domain of CD19 was bound to a NiNTA-lipid incorporated into the supported lipid bilayers . Macrophages expressing a CAR-P with the Megf10 ( CAR-PMegf10 ) or FcRɣ ( CAR-PFcRɣ ) intracellular domain promoted significant engulfment of CD19 beads compared to macrophages with no CAR ( Figure 1b , c , Figure 1—video 1 ) . Macrophages expressing CAR-PBai1 , CAR-PMerTK , and the adhesion-only CAR-PGFP did not bind the CD19 beads even though these CAR-Ps are present at the cell surface ( Figure 1b , c , Figure 1—figure supplement 1 ) . To confirm that the CAR-P was a viable strategy for redirecting primary macrophages , we expressed the CAR-PFcRɣ in primary murine bone marrow derived macrophages and found that these transfected primary cells also were able to trigger engulfment of CD19 beads ( Figure 1d ) . Next we asked if the CAR-P strategy could target a different antigen . Because CAR-PMegf10 performed well in our initial screen ( Figure 1a ) , we developed ɑCD22 CAR-PMegf10 using a previously developed ɑCD22 antibody fragment ( Xiao et al . , 2009; Haso et al . , 2013 ) . Consistent with our results using ɑCD19-based CARs , ɑCD22 CAR-PMegf10 promoted engulfment of CD22 beads ( Figure 2a ) . To confirm antigen specificity of CAR-P , we incubated ɑCD19 CAR-PMegf10 macrophages with CD22 beads , and ɑCD22 CAR-PMegf10 macrophages with CD19 beads . CD19 beads were not eaten by ɑCD22 CAR-PMegf10 macrophages , and CD22 beads were not eaten by ɑCD19 CAR-PMegf10 macrophages ( Figure 2a ) . These data indicate that CAR-PMegf10 specifically triggers engulfment in response to the target ligand and that the CAR-P strategy is able to target multiple cancer antigens . To further define the capabilities of the CAR-P , we assessed the capacity of CAR-P-expressing macrophages to engulf variably sized targets . We found that CAR-PMegf10 was able to trigger specific engulfment of beads ranging from 2 . 5 µm to 20 µm in diameter , with higher specificity above background engulfment being demonstrated for the larger beads ( Figure 2b ) . The high background in this assay is due to heterogeneity in the bilayers on beads purchased from a different manufacturer ( Corpuscular ) than previous assays . For the 10 µm bead condition , we also tested the phagocytic efficiency of beads containing the endogenous Megf10 ligand , phosphatidylserine . We found that CAR-PMegf10 macrophages engulfed CD19 beads and beads containing 10% phosphatidylserine and the adhesion molecule ICAM-1 at a similar frequency ( Figure 2b ) . This indicates that the CAR-P is comparably efficient to the endogenous system . To determine if the CAR-PMegf10 initiates active signaling at the synapse between the macrophage and target , we stained for phosphotyrosine . Macrophages expressing CAR-PMegf10 exhibited an increase in phosphotyrosine at the synapse , while macrophages expressing CAR-PGFP did not show this enrichment ( Figure 3a ) . Consistent with previous reports , we found that F-actin also was enriched at the cell bead synapse ( Figure 3—figure supplement 1 ) . This result suggests that CAR-PMegf10 initiates engulfment through a localized signaling cascade involving tyrosine phosphorylation . Both successful CAR-P intracellular domains ( from FcRɣ and Megf10 ) have cytosolic Immunoreceptor Tyrosine-based Activation Motifs ( ITAMs ) that are phosphorylated by Src family kinases . Based on this observation , we hypothesized that the expression of an alternate ITAM-containing receptor might initiate phagocytosis when expressed in macrophages . The CD3ζ subunit of the T cell receptor contains three ITAM motifs . To test if the CD3ζ chain was able to activate phagocytic signaling , we transduced macrophages with the first generation CAR-T ( Figure 3b ) . The CAR-T was able to trigger engulfment of CD19 beads to a comparable extent as CAR-PMegf10 ( Figure 3c ) . In T cells , phosphorylated ITAMs in CD3ζ bind to tandem SH2 domains ( tSH2 ) in the kinase ZAP70 . Zap70 is not expressed in macrophages , but Syk , a phagocytic signaling effector and tSH2 domain containing protein , is expressed at high levels ( Andreu et al . , 2017 ) . Previous work suggested that Syk kinase can also bind to the CD3ζ ITAMs ( Bu et al . , 1995 ) , indicating that the CAR-T may promote engulfment through a similar mechanism as CAR-PFcRɣ . To quantitatively compare the interaction between SyktSH2 and CD3ζ or FcRɣ in a membrane proximal system recapitulating physiological geometry , we a used liposome-based assay ( Figure 3d [Hui and Vale , 2014] ) . In this system , His10-CD3ζ and His10-Lck ( the kinase that phosphorylates CD3ζ ) are bound to a liposome via NiNTA-lipids and the binding of labeled tandem SH2 domains to phosphorylated CD3ζ was measured using fluorescence quenching . Our results show that Syk-tSH2 binds the CD3ζ and FcRɣ with comparable affinity ( ~15 nM and ~30 nM respectively , Figure 3d ) . Collectively , these results demonstrate that the TCR CD3ζ chain can promote phagocytosis in a CAR-P , likely through the recruitment of Syk kinase . We next sought to program engulfment towards a cellular target . We incubated the CAR-PMegf10 and CAR-PFcRɣ macrophages with cancerous Raji B cells that express high levels of endogenous CD19 . Strikingly , the majority of CAR-P-expressing macrophages internalized bites of the target cell ( Figure 4a , Figure 4—video 1 , 78% of CAR-PMegf10 and 85% of CAR-PFcRɣ macrophages internalized bites within 90 min ) . The biting phenotype resembles trogocytosis , or nibbling of live cells , which has been reported previously in immune cells ( Joly and Hudrisier , 2003 ) . This process was dependent on the ITAM-bearing intracellular signaling domain , as removing the signaling domain ( CAR-PGFP ) dramatically reduced trogocytosis ( Figure 4a ) . Enrichment of phosphotyrosine at the cell-cell synapse further supports active signaling initiating trogocytosis ( Figure 4—figure supplement 1 ) . The CAR-P module also was able to induce trogocytosis in non-professional phagocytes , human NIH 3T3 fibroblast cells ( Figure 4—figure supplement 2 ) . This suggests that the CAR-P can promote cancer antigen-dependent engulfment by both professional and non-professional phagocytes . We next focused on engineering strategies to engulf whole human cancer cells . We observed that macrophages expressing the CAR-PMegf10 or CAR-PFcRɣ were capable of engulfing whole Raji B cells ( 2 cancer cells eaten per 100 macrophages in a 4–8 hr window for both CAR-PMegf10 or CAR-PFcRɣ , Figure 4b , e , Figure 4—video 2 ) . Whole cell engulfment was infrequent but trogocytosis was robust , suggesting that productive macrophage target interactions were frequently insufficient to trigger whole cell engulfment . To determine if whole cell eating could be enhanced by further opsonization of CD19 , we opsonized Raji B cells with a mouse IgG2a anti-CD19 antibody . While addition of this antibody did not trigger additional whole cell internalization , blockade of the ‘don’t eat me’ signal CD47 using the mouse IgG1 anti-human B6H12 clone resulted in a 2 . 5 fold increase of whole cell eating of opsonized Raji B cells ( Figure 4—figure supplement 3 ) . Both endogenous FcR recognition of the anti-CD47 antibody and blockade of CD47 signaling may contribute to this effect . To develop a receptor to enhance whole cell eating , we hypothesized that combining signaling motifs in a tandem array might increase the frequency of whole cell engulfment by specifically recruiting effectors required for the engulfment of large targets . Previous work demonstrated that PI3K signaling is important for internalization of large targets ( Schlam et al . , 2015 ) . To increase PI3K recruitment to the CAR-P , we fused the portion of the CD19 cytoplasmic domain ( amino acids 500 to 534 ) that recruits the p85 subunit of PI3K to the CAR-PFcRɣ creating a ‘tandem’ CAR ( CAR-Ptandem , Figure 4c ) ( Tuveson et al . , 1993; Brooks et al . , 2004 ) . A CAR-P containing the p85 recruitment motif alone ( CAR-PPI3K ) was able to induce some whole cell engulfment , comparable to the CAR-PFcRɣ ( Figure 4e ) . Expression of CAR-Ptandem tripled the ability of macrophages to ingest whole cells compared to CAR-PGFP ( 6 cancer cells eaten per 100 macrophages , Figure 4d , e , Figure 4—video 3 ) . These data indicate that assembling an array of motifs designed to recruit distinct phagocytic effectors can increase CAR-P activity towards whole cells . To determine if the combination of whole cell eating and trogocytosis was sufficient to drive a noticeable reduction in cancer cell number , we incubated CAR-P macrophages with Raji B cells for two days . After 44 hr of co-culture , we found that CAR-P macrophages significantly reduced the number of Raji cells ( Figure 4f ) . Although the CAR-Ptandem was much more efficient at whole cell eating , the CAR-PFcRɣ performed nearly as well at eliminating Rajis . Importantly , our assay does not distinguish between whole cell engulfment or death following trogocytosis , so it is possible both CAR-P activities are contributing to Raji death rates . Overall , these data suggest that the CAR-P is a successful strategy for directing macrophages towards cancer targets , and can initiate whole cell eating and trogocytosis leading to cancer cell elimination . In summary , we engineered phagocytes that recognize and ingest targets through specific antibody-mediated interactions . This strategy can be directed towards multiple extracellular ligands ( CD19 and CD22 ) and can be used with several intracellular signaling domains that contain ITAM motifs ( Megf10 , FcRɣ , and CD3ζ ) . Previous work has suggested that spatial segregation between Src-family kinases and an inhibitory phosphatase , driven by receptor ligation , is sufficient to trigger signaling by the T cell receptor ( Davis and van der Merwe , 2006; James and Vale , 2012 ) and FcR ( Freeman et al . , 2016 ) . The CAR-Ps that we have developed may similarly convert receptor-ligand binding into receptor phosphorylation of ITAM domains through partitioning of kinases and phosphatases at the membrane-membrane interface . Further development of CAR-Ps could be useful on several therapeutic fronts . Targeting of tumor cells by macrophages has been suggested to cause tumor cell killing ( Jaiswal et al . , 2009; Majeti et al . , 2009; Chao et al . , 2010; Jadus et al . , 1996 ) , either through directly engulfing cancer cells or by stimulating antigen presentation and a T cell-mediated response ( Liu et al . , 2015; Tseng et al . , 2013 ) . Inhibition of the CD47-SIRPA ‘Don’t eat me’ signaling pathway has also been shown to result in engulfment of cancer cells ( Chen et al . , 2017; Gardai et al . , 2005; Jaiswal et al . , 2009; Majeti et al . , 2009; Chao et al . , 2010 ) . A recent study suggests that CD47 inhibition is most effective when combined with a positive signal to promote target engulfment , which raises the possibility of combining CAR-P expression with CD47 or SIRPA inhibition for an additive effect ( Alvey et al . , 2017 ) . Although we were able to increase whole cell engulfment by recruiting the activating subunit of PI3K to the phagocytic synapse , the engulfment of larger 20 micron beads was more frequent than the engulfment of whole cells . We hypothesize that this is due to differing physical properties of the engulfment target . Specifically , increased target stiffness has been shown to promote engulfment , suggesting that manipulating the physical properties of the engulfment target could also be a potential strategy for increasing CAR-P efficiency ( Beningo and Wang , 2002; Cross et al . , 2007 ) . While the CAR-P can engulf whole , viable cancer cells , the ingestion of a piece of the target cell is more common . Trogocytosis , or nibbling of living cells , has also been described in immune cells ( Harshyne et al . , 2003; Harshyne et al . , 2001; Kao et al . , 2006; Joly and Hudrisier , 2003; Batista et al . , 2001 ) and brain-eating amoebae ( Ralston et al . , 2014 ) . In vivo studies also have shown that endogenous dendritic cell populations ingest bites of live tumor cells , contributing to presentation of cancer neo-antigen ( Harshyne et al . , 2001; Harshyne et al . , 2003 ) . Although we were able to use the CAR-P to induce trogocytosis in dendritic cells , we were not able to detect robust cross presentation of the model antigen ovalbumin ( Figure 4—figure supplement 4 ) . Thus , although using CAR-Ps to enhance cross presentation of cancer antigen is an intriguing future avenue , such a strategy would likely require more optimization of the dendritic cell subset employed or the CAR-P receptor itself . Overall , our study demonstrates that the CAR approach is transferrable to biological processes beyond T cell activation and that the expression of an engineered receptor in phagocytic cells is sufficient to promote specific engulfment and elimination of cancer cells .
Detailed information for all constructs can be found in the Key resources table . This file includes the following information for all receptors developed in this study: signal peptide , extracellular antibody fragment , stalk/transmembrane domain , and cytosolic tail including appropriate accession numbers . Antibodies used in this study are described in Supplemental Excel File 1 , ‘Antibodies’ tab . J774A . 1 macrophages and NIH 3T3 fibroblasts were obtained from the UCSF cell culture facility and cultured in DMEM ( Gibco , Catalog #11965–092 ) supplemented with 1 x Pen-Strep-Glutamine ( Corning , Catalog #30–009 Cl ) and 10% fetal bovine serum ( FBS ) ( Atlanta Biologicals , Catalog #S11150H ) . Raji B cells were obtained from J . Blau ( McManus lab , UCSF ) and cultured in RPMI ( Gibco , Catalog #11875–093 ) supplemented with 1 x Pen-Strep-Glutamine ( Corning , Catalog #30–009 Cl ) , 10% FBS ( Atlanta Biologicals , Catalog #S11150H ) , 10 mM HEPES ( Gibco , Catalog #1530080 ) , and 5 µM 2-Mercaptoethanol ( Sigma , Catalog #M6250-100mL ) . All cell lines used in this study were tested for Mycoplasma at least once per month using the Lonza MycoAlert Detection Kit ( Lonza , Catalog# LT07-318 ) and control set ( Lonza , Catalog #LT07-518 ) . Lentiviral infection was used to stably express CAR-P constructs in all cell types . Lentivirus was produced by HEK293T cells transfected with pMD2 . G ( a gift from Didier Tronon , Addgene plasmid # 12259 containing the VSV-G envelope protein ) , pCMV-dR8 . 91 ( since replaced by second generation compatible pCMV-dR8 . 2 , Addgene plasmid #8455 ) , and a lentiviral backbone vector containing the construct of interest ( derived from pHRSIN-CSGW ) using lipofectamine LTX ( Invitrogen , Catalog # 15338–100 ) . The media on the HEK293T cells was replaced with fresh media 8–16 hr post transfection to remove transfection reagent . At 50–72 hr post-transfection , the lentiviral media was filtered with a 0 . 45 µm filter and concentrated by centrifugation at 8000 x g for 4 hr or overnight . The concentrated supernatant was applied directly to ~0 . 5×106 NIH 3T3 cells in 2 ml of fresh media . For J77A4 . 1 macrophages and Raji B cells , the concentrated supernatant was mixed with 2 mls of media and 2 µg lipofectamine ( Invitrogen , Catalog # 18324–012 ) and added to the cells . The cells were spun at 2200 x g for 45 min at 37°C . Cells were analyzed a minimum of 72 hr later . Chloroform-suspended lipids were mixed in the following molar ratio using clean glasstight Hamilton syringes ( Hamilton , Catalog #8 1100 ) : 97% POPC ( Avanti , Catalog # 850457 ) , 2% Ni2+-DGS-NTA ( Avanti , Catalog # 790404 ) , 0 . 5% PEG5000-PE ( Avanti , Catalog # 880230 , and 0 . 5% atto390-DOPE ( ATTO-TEC GmbH , Catalog # AD 390–161 ) . Lipid mixes were dried under argon and desiccated overnight under foil . Dried lipids were resuspended in 1 ml tissue-culture grade PBS , pH7 . 2 ( Gibco , Catalog # 20012050 ) , and stored under argon gas . Small unilamellar vesicles were formed by five freeze-thaw cycles followed by 2 × 5 min of bath sonication ( Bioruptor Pico , Diagenode ) , and cleared by ultracentrifugation ( TLA120 . 1 rotor , 35 , 000 rpm / 53 , 227 x g , 35 min , 4°C ) or by 33 freeze thaw cycles . Lipid mixes were used immediately for form bilayers or shock frozen in liquid nitrogen and stored under argon at −80°C . To form bilayers on silica beads , 6 × 108 5 µm silica microspheres ( 10% solids , Bangs Labs , Catalog # SS05N ) were washed 2x in water , and 2x in PBS by sequential suspension in water and spinning at 800 rcf , followed by decanting . Cleaned beads were resuspended in 150 µl tissue-culture grade PBS , pH7 . 2 ( Gibco , Catalog # 20012050 ) and briefly vortexed . 30 µl cleared SUVs prepared as above as a 10 mM stock were added to bead suspension for a 2 mM final SUV concentration . Beads were vortexed for 10 s , covered in foil , and rotated for 30 min at room temperature to form bilayers . Bilayer-coated beads were washed 3x in PBS by sequential centrifugation at 800 rcf and decanting . Beads were resuspended in PBS + 0 . 1% w/v BSA for blocking for 15 min rotating at room temperature under foil . 10 nM final concentration of CD19his8 ( Sino Biological , Catalog # 11880H08H50 ) or CD22his8 ( Sino Biological , Catalog # 11958H08H50 ) protein were added to blocked beads and proteins were allowed to bind during a 45 min incubation rotating under foil at room temperature . Beads were washed 3x in PBS + 0 . 1% w/v BSA by sequential centrifugation at 300 rcf and decanting . Beads were resuspended in 120 µl PBS + 0 . 1% w/v BSA . Prior building bilayers on Silica beads ranging from 2 . 5 µm-20 µm in diameter ( Microspheres-Nanospheres , Catalog# C-SIO-2 . 5 , 5 , 10 , 15 , 20 ) , beads were RCA cleaned as follows: beads were pelleted at 2000 x g in low retention tubes ( Eppendorf , Catalog #022431081 ) and resuspended in acetone . Resuspended beads were sonicated for 60 min in a bath sonicator . Rinse and sonication were repeated in ethanol . Finally , rinse and sonication were repeated in water . Beads were then washed 2x in water to remove all traces of ethanol and left in a small volume after decanting . All further steps were performed in a 70–80°C water bath prepared in a fume hood . Proper Personal Protective Equipment ( PPE ) was worn throughout the RCA cleaning protocol . Washed beads were added to 3 ml of hot 1 . 5 M KOH in a clean glass vial suspended in the water bath described above . 1 ml 30% H2O2 to bead solution and allowed to react for 10 min . Washed beads were cooled on ice , pelleted at 2000xg and rinsed 5x in ultrapure water . Used cleaning solution was saved for disposal by Environmental Health and Safety ( EH and S ) . Cleaned beads were resuspended in 240 µl tissue-culture grade PBS , pH7 . 2 ( Gibco , Catalog # 20012050 ) and briefly vortexed . The lipid mix used in this assay differed slightly from above . Here a mix of 93 . 5% POPC ( Avanti , Catalog # 850457 ) , 5% Ni2+-DGS-NTA ( Avanti , Catalog # 790404 ) , 1% PEG5000-PE ( Avanti , Catalog # 880230 , and 0 . 5% atto390-DOPE ( ATTO-TEC GmbH , Catalog # AD 390–161 . Bilayers were built and proteins coupled as described above . The concentration of CD19 was scaled appropriately to account for the increased surface area of the larger beads . 12 to 16 hr prior to imaging , 2 . 5 × 104 J774A . 1 macrophages expressing the appropriate CAR-P or control construct were plated in a 96-well glass bottom MatriPlate ( Brooks , Catalog # MGB096-1-2-LG-L ) . To assess engulfment , 0 . 5 × 106 CD19 or CD22-ligated beads were added to each well . Engulfment was allowed to proceed for 45 min at 37°C incubator with CO2 . Cells were then imaged as described below . On the day of imaging 0 . 5 × 106 NIH 3T3 fibroblasts , dendritic cells or macrophages and 1 . 5 million Raji B cells were combined in a 1 . 5 ml eppendorf tube and pelleted by centrifugation ( 800 rpm / 68 x g ) for 5 min at room temperature . Culture media was decanted to ~100 µl volume and cells were gently resuspended , and allowed to interact in the small volume for 60 min in a 37°C incubator with CO2 . After incubation cells and beads were diluted to a final volume of 1000 µl and 300 µl of this co-culture plated for imaging in a 96-well glass bottom MatriPlate ( Brooks , Catalog # MGB096-1-2-LG-L ) , and imaged as described below . 20 , 000 J774A . 1 macrophages were plated into 96-well glass bottom MatriPlate ( Brooks , Catalog # MGB096-1-2-LG-L ) in a final volume of 300 µl complete DMEM ( Gibco , Catalog #11965–092 ) supplemented with 1 x Pen-Strep-Glutamine ( Corning , Catalog #30–009 Cl ) and 10% fetal bovine serum ( FBS ) ( Atlanta Biologicals , Catalog #S11150H ) . 52 hr prior to reading the assay macrophages were stimulated with 500 ng/ml LPS ( Sigma , Catalog # L4516 ) . 44 hr prior to imaging LPS was removed by three sequential gentle washes . After LPS removal 10 , 000 Rajis expressing mCherry-CAAX were added to the well containing stimulated macrophages . The co-culture was incubated for 44 hr in a 37°C tissue culture incubator with 5% CO2 . After 44 hr , the remaining number of Raji B cells remaining was analyzed by FACS as follows: 10 , 000 counting beads were added to the well immediately prior to reading and the cell-counting bead mixture was harvested by pipetting up and down 8x with a p200 pipet . The assay was read on an LSRII ( BD Biosciences ) and Rajis were identified by the presence of mCherry fluorescence . Bone marrow derived macrophages ( BMDMs ) were produced as previously described ( Weischenfeldt and Porse , 2008 ) , except that L-929 conditioned media was replaced with purified 25 ng/ml M-CSF ( Peprotech , Catalog # 315–02 ) . The BMDMs were lentivirally infected with concentrated lentivirus after 5 days of differentiation . Differentiation was confirmed by F4/80 staining on day seven and found to be >95% efficient for each replicate . Phagocytosis was measured on day nine in imaging media lacking M-CSF . To produce CAR-P expressing dendritic cells , bone marrow-derived hematopoietic stem cells were lentivirally infected immediately after harvest by spinning with concentrated lentivirus in GMCSF-containing media ( IMDM supplemented with 10% FBS and PSG ) on retronectin ( Clontech , Catalog # T100A ) -coated plates at 2200 x g for 45 min at 37°C . Dendritic cells were produced as previously described ( Mayordomo et al . , 1995 ) by culturing bone marrow cells for 8–11 days with GMCSF . IL-4 was added 2–3 days before use . Efficient differentiation into CD11c + dendritic cells was verified by FACS , revealing ≥95% APC-CD11c + cells ( Biolegend , Catalog #N418 ) . The ability of CAR-P to stimulate OTI T cell proliferation was tested using the co-culture assay shown as a schematic in Figure 4—figure supplement 4 and described previously ( Roberts et al . , 2016 ) . 10 , 000 CAR-P transduced CD11c + dendritic cells transduced and differentiated as above were plated in U bottom 96 well dishes ( Falcon , Catalog #353077 ) and stimulated with 1 ug/ml LPS . 12 hr after LPS stimulation , 40 , 000 Raji B cells expressing soluble cytosolic ovalbumin ( Raji B-OVA ) were added to the culture . 24 hr after Raji B-OVA cell addition , 50 , 000 OTI CD8 + T cells isolated from lymph nodes of OTI TCR transgenic mice using a CD8 +T cell purification kit ( Stemcell , Catalog #19853 ) and labeled with e670 proliferation dye ( Thermo , Catalog #65-0840-85 ) were added . 72 hr after OTI addition the percent of OTI cells divided was measured by eFluor670 signal using flow cytometry . All imaging in this study was performed using a spinning disk confocal microscope with environmental control ( Nikon Ti-Eclipse inverted microscope with a Yokogawa spinning disk unit ) . For bead internalization assays , images were acquired using a 40 × 0 . 95 N/A air objective and unbiased live image acquisition was performed using the High Content Screening ( HCS ) Site Generator plugin in µManager3 . Other images were acquired using either a 100 × 1 . 49 N/A oil immersion objective . All images were acquired using an Andor iXon EM-CCD camera . The open source µManager software package was used to control the microscope and acquire all images3 . 20 , 000 J774A . 1 macrophages were plated into 96-well glass bottom MatriPlate ( Brooks , Catalog # MGB096-1-2-LG-L ) . Four hours prior to imaging , the macrophages were stimulated with 500 ng/ml LPS ( Sigma , Catalog # L4516 ) . Immediately prior to imaging the LPS-containing media was replaced with Fluobrite DMEM ( ThermoFisher Scientific , Catalog # A1896701 ) containing 10% FBS . 40 , 000 Raji cells were added to the macrophages and the co-culture was imaged at 5 min intervals for 12 hr . For the antibody experiments , macrophages were washed into DMEM minus phenol red ( A14430-01 ) containing 10% FBS just prior to addition of 40 , 000 Raji cells . Where indicated antibody was added to a final concentration of 20 µg/ml immediately after Raji cell addition and prior to imaging to limit antibody internalization . Because cells moved in and out of the field of view , we selected the cells present after 8 hr of imaging and quantified their B cell eating if they could be followed for four hours or more . Time-lapse analysis was essential to ensure that the B cell appeared viable prior to engulfment by the macrophage . Engulfment of B cells with an apoptotic morphology was not counted as a whole cell eating event . During live cell image acquisition GFP-positive J774A . 1 macrophages or NIH 3T3 cells were selected by the presence of GFP signal . A full z-stack comprising the entire cell was captured using 1 µm steps . All z sections were then manually inspected for internalized Raji B cell material . Cells containing one or more bites of fully internalized Raji B cell material >1 µm in diameter were scored as positive . Experiments were carried out as previously described2 . Briefly , proteins were purified using a bacterial expression system . All protein components ( 1 mg/ml BSA , 100 nM tSH2-Syk SNAP-505 , 0 to 500 nM His10-CD3ζ or His10-FcRɣ intracellular chain , and 7 . 2 nM His10-Lck Y505F ) were mixed into kinase buffer ( 50 mM HEPES-NaOH pH 6 . 8 , 150 mM NaCl , 10 mM MgCl2 , and 1 mM TCEP ) . Liposomes prepared at the following molar ratios: 74 . 5% POPC ( Avanti , Catalog # 850457C ) , 10% DOGS-NTA ( Nickel ) ( Avanti , Catalog # 790404C , 0 . 5% Rhodamine PE ( Avanti , Catalog # 810150C ) , and 15% DOPS ( Avanti , Catalog # 840035C ) were added and the mixture was incubated for 40–60 min at room temperature , during which the SNAP-505 fluorescence was monitored at 8 s intervals with 504 nm excitation and 540 nm emission . 1 mM ATP was then injected to trigger Lck mediated phosphorylation of CD3ζ or FcRɣ . Injection was followed by 5 s of automatic shaking of the plate , and the fluorescence was further monitored at 8 s intervals for at least 1 hr . Data were normalized by setting the average fluorescence value of the last 10 data points before ATP addition as 100% and background fluorescence as 0% . The final extent of fluorescence quenching ( % fluorescence change ) at each concentration of receptor was determined using the average of the last 20 data points after ensuring fully equilibrated binding . Nine reactions containing increasing concentrations of CD3ζ and nine reactions containing increasing concentrations of FcRɣ were run in parallel . The final % fluorescence change was plotted against FcRɣ or CD3ζ concentration . The apparent dissociation constants ( Kd ) of tSH2-Syk to FcRɣ and CD3ζ were calculated by fitting the data with Graphpad Prism 6 . 0 , using the ‘one site specific binding’ model . The intracellular portion of the FcR γ-chain ( aa 45–85 , Human FcRɣ , Uniprot FCERG_HUMAN ) was cloned into a modified pET28a vector containing a His10 upstream to the multiple cloning site using BamHI and EcoRI . The intracellular portion of CD3ζ ( aa 52–164 , Human CD3ζ , Uniprot CD3Z_HUMAN ) was also cloned into the His10 modified pET28a vector . A Lys-Cys-Lys-Lys sequence , originally present for fluorescent labeling , is also present between His10 and CD3ζ in this construct . SNAP-tSH2Syk ( aa 1–262 ) was cloned into a pGEX6 vector using BamHI and EcoRI . His10-CD3ζ , His10-FcR γ-chain , and GST-SNAP-tSH2Syk were bacterially expressed in BL21 ( DE3 ) RIPL strain of Escherichia coli as described previously2 . His10-Lck Y505F was expressed in SF9 cells using the Bac-to-Bac baculovirus system as described previously2 . All cells were lysed in an Avestin Emulsiflex system . His10 proteins were purified by using Ni-NTA agarose ( Qiagen , Catalog # 30230 ) and GST-SNAP-tSH2Syk was purified by using glutathione-Sepharose beads ( GE Healthcare , Catalog # 17075601 ) as described previously2 . Soluble SNAP-tSH2 Syk was generated by cleaving the GST moiety via the PreScission Protease at 4°C overnight . All proteins were subjected to gel-filtration chromatography using a Superdex 200 10/300 GL column ( GE Healthcare , Catalog # 17517501 ) in HEPES-buffered saline ( HBS ) containing 50 mM HEPES-NaOH ( pH 6 . 8 for His10-CD3ζ , His10-FcR γ-chain , and GST-SNAP-tSH2Syk and pH 7 . 4 for His10-Lck Y505F ) , 150 mM NaCl , 5% glycerol , and 1 mM TCEP . The monomer fractions were pooled , frozen in liquid nitrogen and stored at −80°C . All gel-filtered proteins were quantified by SDS-PAGE and Coomassie staining , using BSA as a standard . To prepare fluorescently labeled tSH2 Syk , 10 uM SNAP-tSH2 Syk was incubated at a 1:2 ratio with SNAP-Cell 505-Star ( NEB , Catalog # S9103S ) overnight at 4°C and run over a PD MiniTrap G-25 ( GE Healthcare , Catalog # 28-9225-29 AB ) column to eliminate excess dye . To fix and stain preparations described above in bead and bites assays for quantifying enrichment of phosphotyrosine staining , half the media ( ~150 µl ) was gently removed from the imaging well and replaced with 150 µl 6 . 4% paraformaldehyde solution ( prepared from 32% stock , Electron Microscopy Sciences , Catalog # 50980495 ) in tissue culture grade PBS , pH7 . 2 ( Gibco , Catalog # 20012050 ) . Cells were fixed for 15 min in a 37°C incubator with CO2 . After fixation cells were washed 2x with PBS and permeabilized/blocked for 60 min at room temperature in freshly prepared , filter sterilized PBS + 5% FBS+0 . 1% w/v saponin ( PFS solution ) . After permeabilization , cells were washed 2 × 3 min with PFS solution . Following block , cells were incubated with 1:100 dilution of mouse anti-phosphotyrosine ( pTyr ) antibody to stain pan-pTyr ( Santa Cruz , Catalog # PY20 ) diluted in PFS solution in the dark for 60 min at room temperature then washed 3 × 5 min in PFS solution . Washed cells were incubated with a 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibody ( Thermo/Molecular Probes , Catalog # A21236 ) in PFS solution in the dark for 60 min at room temperature . Wells were then washed 3 × 5 min in PFS solution . Cells were covered in 200 µl PBS . If not imaged immediately samples were wrapped in parafilm and foil and stored at 4°C prior to microscopy . Phosphotyrosine enrichment at the synapse was calculated by dividing the mean Alexa Fluor 647 signal of a 5 pixel linescan at the synapse with bead or cell by a 5 pixel linescan on the cortex . For phalloidin staining , cells were fixed with 4% PFA for 15 min at room temperature , blocked and permeabilized with 5% BSA in TBS with 0 . 5% triton X overnight , and incubated with AlexaFluor 647 Phalloidin ( Thermo/Molecular Probes , Catalog # A22284 ) for 20 min . Cells were then washed with PBS , imaged and quantified using the method described above . Each data point represents a single cell , and the graphs reflect pooled results from three biological replicates . To fix and stain preparations described above for ovalbumin staining , half the media ( ~150 µl ) was gently removed from the imaging well and replaced with 150 µl 8% paraformaldehyde solution ( prepared from 32% stock , Electron Microscopy Sciences , Catalog # 50980495 ) in tissue culture grade PBS , pH7 . 2 ( Gibco , Catalog # 20012050 ) . Cells were fixed for 10 min in a 37°C incubator with CO2 . After fixation cells were washed 2x with PBS and permeabilized/blocked for 60 min at room temperature in freshly prepared , filter sterilized PBS + 0 . 1% w/v casein +0 . 1% w/v saponin ( PCS solution ) . After permeabilization , cells were washed 1 × 3 min with PCS solution and blocked for 1 hr at room temperature in PCS . Following block , cells were incubated with 1:100 dilution of rabbit anti-ovalbumin ( OVA ) antibody to stain OVA ( Thermo/Pierce , Catalog # PA1-196 ) diluted in PCS solution overnight at 4°C . Washed cells were incubated with a 1:200 dilution of goat anti-rabbit Alexa Fluor 647 antibody ( Thermo/Molecular Probes , Catalog # A21235 ) and 3 . 3 nM 488 phallodin ( dissolved at 6 . 6 µM in methanol ) in PCS solution in the dark for 60 min at room temperature . Wells were then washed 3 × 5 min in PCS solution . Cells were covered in 200 µl PBS and immediately imaged . Ovalbumin signal was quantified as the corrected total cell fluorescence ( CTCF ) . CTCF = Integrated Density – Area of Selected Cell * Mean Fluorescence of 3 Background Readings . Each data point represents a single cell , and the graphs reflect pooled results from three biological replicates . All image quantification was done on raw , unedited images . All images in figures were first analyzed in ImageJ , where a single Z-slice at the center of the cell was extracted . The image intensities were scaled to enhance contrast and cropped in Photoshop . For movies , background was subtracted in Fiji using a rolling ball radius of 50 μm and bleach corrected using the Histogram Matching plug in . All statistical analysis was performed in Prism 6 . 0 ( GraphPad , Inc . ) . The statistical test used is indicated in each figure legend . Error bars throughout the paper denote 95% confidence intervals of the mean . *** indicates p<0 . 0001; ** indicates p<0 . 001 and * indicates p<0 . 01 .
Chimeric antigen receptors ( CARs ) are synthetic receptors that reprogram T cells to kill cancer . The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial . Here , we engineered a family of Chimeric Antigen Receptors for Phagocytosis ( CAR-Ps ) that direct macrophages to engulf specific targets , including cancer cells . CAR-Ps consist of an extracellular antibody fragment , which can be modified to direct CAR-P activity towards specific antigens . By screening a panel of engulfment receptor intracellular domains , we found that the cytosolic domains from Megf10 and FcRɣ robustly triggered engulfment independently of their native extracellular domain . We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells . Addition of a tandem PI3K recruitment domain increased cancer cell engulfment . Finally , we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40% .
Our immune system constantly patrols our body , looking to eliminate cancerous cells and harmful microbes . It can spot these threats because it recognizes certain signals at the surface of dangerous cells . However , cancer cells often find ways to ‘hide’ from our immune system . Chimeric antigen receptors , or CARs , are receptors designed in a laboratory to attach to specific proteins that are found on a cancer cell . These receptors tell immune cells , such as T cells , to attack cancers . T cells that carry CARs are already used to treat people with blood cancers . Yet , these immune cells are not good at penetrating a solid tumor to kill the cells inside , which limits their use . Macrophages are a group of immune cells that can make their way inside tumors and travel to cancers that the rest of the immune system cannot reach . They defend our body by ‘swallowing’ harmful cells . Would it then be possible to use CARs to program macrophages to ‘eat’ cancer cells ? Morrissey , Williamson et al . created a new type of CARs , named CAR-P , and introduced it in macrophages . These cells were then able to recognize and attack beads covered in proteins found on cancer cells . The modified macrophages could also limit the growth of live cancer cells in a dish by ‘biting’ and even ‘eating’ them . While these results are promising in the laboratory , the next step is to test whether these reprogrammed macrophages can recognize and fight cancers in living animals .