Immune-stimulating Antibody Conjugates Elicit Robust Myeloid Activation And Durable Anti-tumor Immunity(Part 2)

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Discussion:

The results presented here provide compelling evidence supporting ISACs as a novel technology for tumor immunotherapy. In vitro modeling of ISACs demonstrated their ability to activate primary human myeloid APCs in an FcγR and TLR-dependent manner and to enhance antigen cross-presentation using the murine OVA model system. Activation through upregulation of costimulatory molecule CD40 was not only observed in vitro but was also seen on tumor-residing myeloid APCs in both xenograft and syngeneic tumor models. One of the striking findings of this study was that the covalent attachment of a TLR agonist to a tumor-targeted monoclonal antibody in the form of an ISAC altered the immunostimulatory outcome and overall efficacy that is typically anticipated with TLR agonists. A comparison of local delivery of trastuzumab combined with T785 to systemic delivery of the T785-ISAC demonstrated that only the ISAC was successful in reducing tumor burden. Local delivery of the mixture provided no benefit in the HCC1954 tumor xenograft model, with comparable effects to trastuzumab alone. This finding supports that not only the local delivery but also the simultaneous delivery of the antibody and TLR agonist is essential for the therapeutic efficacy observed with the ISAC. Mechanistic data generated in the HCC1954 tumor xenograft model demonstrated the in vivo requirement for FcγR engagement and TLR engagement for efficacy, as both the TLRnull-ISAC and Fc-inactive ISAC failed to control tumor growth. Furthermore, systemic administration of ISACs resulted in sustained anti-tumor efficacy in rat HER2+ syngeneic tumor models and human HER2+ breast cancer xenografts that were resistant to treatment with the unconjugated antibody. HER2 was selected as the target antigen for preclinical in vivo studies because neoadjuvant, adjuvant, and first-line therapy for patients with locally advanced or metastatic breast cancer is dominated by HER2-targeted antibodies that are not designed to stimulate the immune system and approved T cell checkpoint inhibitor blocking antibodies have been minimally effective in these patients. These patients could benefit from ISACs that retain the functionality of the parent antibodies and add significant immunostimulatory potential. As with any immunotherapeutic, the possibility to generate or exacerbate anti-drug antibodies exists and, in the case of the ISAC, may ultimately depend on the immunogenicity of the parent antibody. These assessments are best performed clinically and trastuzumab may be ideally suited for investigation as the reported immunogenicity rate of Herceptin™ in patients is less than 1%. In vivo data demonstrating the requirement for simultaneous delivery of the antibody and adjuvant is further supported by work detailing the intracellular signaling governing ISAC activity through simultaneous FcγR and TLR signaling. While additional studies may further elucidate the intracellular ISAC signaling dynamics, the studies described here highlight the potential signaling advantages of conjugating a TLR agonist to an antibody, as is typically observed when antibodies engage pathogens during protective immunity. CyTOF-based analysis of intracellular signaling in human PBMCs was employed to further understand and differentiate the activity of the mixture and ISAC. The results revealed an amplified signature following ISAC stimulation compared to that elicited by the mixture of individual components, further supporting the potential for enhancement of activity through chemical conjugation of the antibody and TLR agonist. While TLR7 and TLR8-specific potency of ISACs cannot be measured using HEK293 reporter cells, the demonstration of ISAC-induced phosphorylation of IRF-7 suggests active TLR7/8 agonism, as IRF-7 is known to be downstream of TLR7 and TLR8 MyD88-driven signaling 30. The data indicate that the ISAC not only elicits increased signaling of canonical pathways associated with TLR and FcγR pathways but also reduces the pairwise activation threshold required to trigger phosphorylation of downstream signaling proteins such as ribosomal protein S6, a key regulator of protein translation in the cell. High DREMI scores, particularly measured in monocytes as well as in DCs, support the strong conditional dependence induced by stimulation with the ISAC that is not seen with the mixture. This phenomenon may be facilitated by intracellular changes in abundance, localization, and/or recruitment of receptors within the endosome, but further investigation is required to confirm an exact mechanism. Importantly, DREMI analysis identified relationships consistent with known signaling partners as a result of ISAC stimulation. By its known inhibitory role in canonical NF-κB signaling 49, we observed reduced dependency (i.e. DREMI score) between pIRF7 and inhibitor of kappa B (IκB) when myeloid cells were stimulated with ISAC. Collectively, these results support an amplified and sustained intracellular response via relevant signal transduction pathway molecules downstream of ISAC stimulation in addition to the synergy between the TLR and FcγR-related signaling pathways.

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Our results revealed that functional Fc-FcγR interactions are essential for ISAC-mediated activation of myeloid APCs. Native IgG1 Fc, which has the highest affinity for the activating FcγRs of the naturally existing IgG isotypes, was superior to other IgG isotypes with lower affinities for the activating FcγRs. Further enhancement of the Fc-FcγR interaction through fucosylation enhanced potency while diminution of the Fc-FcγR interaction through glycosylation reduced potency. As expected, Syk signaling downstream of FcγR engagement in addition to subsequent TLR agonism was required for ISACs to elicit maximal stimulation. These findings are supported by studies demonstrating a dramatic enhancement of myeloid stimulation following co-stimulation with plate-bound IgG and the TLR agonist PAM3CSK 50. They also suggest that more extensive Fc crosslinking, on the target cell surface or within the in vitro plate setting, may occur, or differential signaling networks are engaged, following ISAC stimulation as opposed to the mixture of components. Translation of the ISAC platform in vivo was first assessed with human HER2+ tumor cell lines in mice that lack fully functional B, T, and NK cells. Remarkably, the trastuzumab ISAC was significantly more effective than trastuzumab across multiple tumors with varying levels of HER2 expression. Furthermore, the data demonstrated the dependence on tumor- targeting for anti-tumor effects of the ISAC, as the isotype-ISAC was unable to induce tumor regression and clearance. While in vitro modeling demonstrated a necessity for TLR and FcγR activity to enable ISAC potency, such models were not successful in measuring the dependence on ISAC antigen-targeting. No differences were measured between targeted and isotype T785-ISACs in plate-based assays with healthy human myeloid APCs co-cultured with tumor cells (data not shown). The lack of target-dependence measured in vitro may be attributed to multiple factors: myeloid APCs are found to upregulate FcγRs on the cell surface following in vitro culture, which may enhance the ability for soluble IgG to bind to FcγR, leading to an amplified signal from the isotype ISAC. Additionally, the cultured system lacks endogenous IgG and additional serum factors present in vivo that may compete for FcγR in vivo and dampen effects seen with an isotype ISAC. Nonetheless, the in vivo models demonstrated the ISAC’s reliance on tumor-targeting for anti-tumor efficacy. Not only did isotype ISACs show little impact on tumor growth in efficacy studies, but they were also found to induce no impact on the genetic level in tumors, as shown in xenograft and syngeneic tumor models. In addition to demonstrating target-dependent efficacy in vivo, the T785-ISAC was tolerated by animals and induced minimal weight loss, demonstrating little sign of off-target effects and adverse events in the mouse. These data further support the ability of a systemically-delivered tumor-targeting ISAC to travel to the tumor and induce a localized pro-inflammatory anti-tumor response.

The importance of myeloid cell-mediated effector functions was demonstrated in cell-depleting studies, in which clodronate depletion of phagocytes ablated the ISAC’s ability to induce tumor clearance. Interestingly, although tumors initially regressed after depletion of Gr1 positive cells, the tumors subsequently grew out. This phenomenon may have occurred as a result of depletion of phagocyte precursors such as monocytes, which may have later differentiated into functional and mature phagocytic cells 51. Further molecular and cellular insights were obtained upon quantifying mRNA levels and changes following ISAC treatment. These data were further corroborated by immunohistochemistry as well as protein quantification, indicating an infiltration of myeloid cells into the tumor along with the production of a pro-inflammatory cytokine TNFα and myeloid chemoattractants CCL2 (MCP-1) and CCL4 (Mip1b).

Figure 2: ISACS elicit distinct signaling patterns in myeloid APCs. (a-d) Freshly isolated human PBMC were stimulated with 1 μM of the rituximab-ISAC or an equimolar equivalent of the mixture in the presence of CD20+ Toledo tumor cells at a 1:1 ratio for 15 minutes. (a) The median arcsinh ratio of ISAC over signaling was measured following mixture stimulation for various phosphoproteins as depicted by a heat map, with yellow indicative of increased signaling and blue indicative of reduced signal. (b) Representative flow cytometry plots for pIRF7 and pRPS6 in monocytes and cDCs. (c) Fold change of signaling induced was computed as the arcsinh ratio over the unstimulated control. IRF7 and ERK1/2 phosphorylation was measured in monocytes and cDCs following ISAC or mixture stimulation. Data are from one experiment with six donors (mean and SEM); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (d) Representative DREVI plots following DREMI analysis with the area under the curve (AUC) and inflection point (IP) denoted. DREVI plots were visually inspected for curve fit and signal-to-noise, and inflection points were calculated for those with proper sigmoidal curve fits (N/A indicates no curve fit).

The receptors for CCL2 and CCL4, CCR2 and CCR5 are expressed on monocytes and macrophages, respectively, suggesting these cell types could be recruited to ISAC-treated tumors. Furthermore, Cxcl9 and Cxcl11 mRNA, which encode T cell-specific chemokines and be expressed by DCs in the TME, were also upregulated by trastuzumab ISAC treatment (Supplemental Figure 23) 52,53. A trastuzumab ISAC with increased potency, using the agonist CL264, exhibited enhanced efficacy in tumor models expressing less HER2. This is important given that patients whose tumors express low HER2 have more limited treatment options and are not eligible for trastuzumab or other trastuzumab-containing therapies. Transient weight loss of about 5-10% and increased systemic secretion of TNFα were observed in animals treated with both the targeted and isotype CL264-ISACs, indicative of possible off-target effects in addition to the robust anti-tumor activity observed. The collective data presented in the xenograft models suggest that ISAC-mediated phagocytosis is an effective mechanism to eliminate tumors, but this mechanism might be enhanced following antigen presentation of tumor neoantigens to T cells in immunocompetent hosts. Toward this end, the ability of ISACs to promote anti-tumor immunity in wild-type mice was investigated with anti-rHER2 ISACs in two different rHER2-expressing syngeneic models (MMC and CT26-rHER2). Both T785 and CL264 containing ISACs were assessed in the syngeneic MMC model given the high antigen expression of rHER2, as both the lower and higher potency ISACs were postulated to be efficacious with high antigen density on the tumor cell. Both rHER2 ISACs led to complete tumor clearance in animals bearing large tumors (350-950 mm3) in the MMC model whereas the unconjugated antibody failed to control tumor growth. Analysis of mRNA transcript levels in the MMC model revealed a dramatic shift in the regulation of many genes at 24 hours following treatment with the targeted rHER2 T785-ISAC relative to the rHER2 antibody or isotype ISAC, including within genes involved in myeloid cell biology, TLR biology, and adaptive immune responses. Increased infiltrates of CD11c- and F4/80-expressing cells were seen by immunohistochemistry, similar to the trend seen in the HCC1954 xenograft tumor model. Furthermore, CD8 T cell infiltration was observed by immunohistochemistry, supporting the generation of an adaptive immune response following ISAC treatment.

In addition to the demonstrated importance of phagocytes in the xenograft model, depletion studies in the MMC syngeneic tumor model revealed a strong dependence on phagocytes, as depletion using clodronate-loaded liposomes halted ISAC-mediated anti-tumor effects. Moreover, the syngeneic studies demonstrated a critical role for T cells in ISAC efficacy. ISAC-driven tumor clearance was heavily dependent on CD8 T cell activity, as depletion of CD8 T cells inhibited anti-tumor efficacy. The data suggest that T cell activity is driven through TLR-mediated activation of ISAC-stimulated myeloid cells. Such requirement for T cells, presumably related to a presentation of tumor-associated antigens by ISAC-stimulated myeloid cells, supports that ISACs likely mediate their anti-tumor activity through at least two mechanisms: phagocytosis and antigen presentation. While phagocytes provide an early anti-tumor response following ISAC treatment, antigen priming of T cells ultimately provides the tumor clearance and durable effects measured in fully immunocompetent models. This combination would be highly desirable for cancer patients as ISAC treatment could lead to robust and durable anti-tumor immunity. To further explore the importance of ISAC-mediated T cell activation, mice cured of CT26- rHER2 tumors following rHER2 CL264-ISAC treatment were rechallenged, after 30 days of being tumor-free, with the non-rHER2 expressing CT26 parental cell line. rHER2-CT26 cured animals were protected from the challenge with CT26 whereas tumor naïve animals were unable to reject tumor growth. Additionally, to show that protection was specific to the CT26 tumor cell line, 4T1 was simultaneously implanted in the contralateral flank of parental CT26 challenged mice. Although CT26 tumors failed to grow in her-CT26 cured mice, 4T1 tumor growth was unencumbered and thus provides evidence that immunity is antigen-specific and dependent upon the adaptive immune response. Furthermore, these studies suggest tumor-targeted ISACs mediate immunological memory not only to the initially targeted tumor antigen but also to untargeted antigens. These data also suggest that myeloid cells may present tumor-associated antigens that drive additional CTL responses. The implications of these findings demonstrate that, even if the targeted tumor antigen is subsequently downregulated or unable to bind to the ISAC, immunological memory has already been invoked and is capable of sustaining a durable response. This response is not limited to the antigen being targeted by the ISAC but also to additional, unidentified, tumor-expressed antigens. Taken together, these data indicate that ISACs elicit qualitatively and quantitatively distinct biology relating to cellular activation and antigen presentation, T cell proliferation, and durable anti-tumor immunity.

Methods and Materials: Antibody Conjugation & Characterization For conjugation of adjuvants to the antibody, adjuvants were first synthesized to include a linker flanked by a reactive group. Conjugates produced using two-step methods were first synthesized through the modification of mAb lysine residues with a heterobifunctional crosslinker SATA. Deprotection of the acetylated thiol exposed a reactive thiol which was then reacted at room temperature for 2-4 hours with an adjuvant-linker flanked by a thio-reactive maleimide. For constructs produced using a single-step conjugation method, TFP esters were then conjugated to an IgG1 antibody. The TFP esters were dissolved in anhydrous DMSO to make a 20 mM stock solution and 5-10 molar equivalents (relative to the antibody) were added to the IgG antibody at 10 mg/mL in PBS. The conjugation reaction was performed at 4-40°C for 2-12 hours. The resulting immunoconjugates were buffer exchanged into PBS (pH 7.4) through desalting (Zeba Columns, Thermo Fisher Scientific) or dialysis to remove excess small molecular weight impurities. The final protein concentration was determined by measuring the absorbance at 280 nm on a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). The yields were >75% based on recovered protein. SEC analysis detected minimal aggregate presence and DAR was determined by LC/MS analysis. The purified ISACs were filtered through a 0.2 μm sterile filter and stored at −20°C until use. HEK Reporter Assay HEK293 reporter cells expressing human TLR7 or human TLR8 were purchased from Invivogen (hub-htlr7 or hub-htlr8) and vendor protocols were followed for cellular propagation and experimentation. Briefly, cells were grown to 80-85% confluence at 5% CO2 in DMEM supplemented with 10% FBS, Zeocin, and Blasticidin. Cells were then seeded in 96-well flat plates at 4x104 cells/well with substrate containing HEK detection medium and immunostimulatory molecules. The activity was measured using a plate reader at 620-655 nm wavelength. Biacore Analysis His-tagged Fc Gamma Receptor (FCGR) proteins were obtained from R&D Systems (FCGR1 (CD64, cat# 1257-FC-050), FCGR2A (CD32a, cat# 1257-FC-050), FCGR2B (CD32b, cat# 1875-FC-050), FCGR3A (CD16a, cat# 4235-FC-050). Binding analysis was performed on a Biacore T200 instrument in HBS-EP+ buffer (GE BR100669) using a CM5 chip (GE BR100530) containing immobilized anti-HIS antibody (HIS Capture kit, GE 28995056). Rituximab or Rituximab-ISAC have injected over flow cells containing captured FCGRs or a reference surface anti-His antibody only. Data was double referenced with the anti-His surface and a buffer-only injection. A kinetic titration method was used and surfaces were regenerated between cycles by injection of 10 mM glycine, pH 1.5. Data were fit using Biacore T200 evaluation software (V3.1) using kinetic fit (1:1) for FCGR1 and FCGR3A (average of 6 runs) and steady-state affinity (1:1) for FCGR2A and FCGR2B (average of 3 runs).

Human Myeloid APC Isolation Myeloid APCs, which predominantly consisted of monocytes (>95%), were isolated from the whole blood of healthy donors (Stanford Blood Center) by density gradient centrifugation using a RosetteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies). Myeloid APCs were further isolated using a negative selection Human Monocyte Enrichment Kit without CD16 depletion (Stem Cell Technologies) to a final purity of >90% as determined by flow cytometry based on CD14, CD16, CD11c, and HLA-DR expression. Toledo Tumor Cell Preparation Toledo cells (ATCC) were cultured in vendor-recommended media and maintained at vendor-recommended cell densities (ATCC). Cells were removed from the culture, washed, and resuspended in PBS with 2% FBS and 2 mM EDTA at 1-10x106 cells/mL. In some cases, cells were subsequently labeled with 2 μM CFSE for 2 minutes and then washed once with RPMI-1640 (Lonza) supplemented with 10% FBS (Atlanta Biologicals) and 100 U/mL Pen/Strep (Lonza). Cells were then fixed in 2% paraformaldehyde and washed three times with PBS before further use. Cellular Activation Assays PBMCs or isolated monocytes were plated at 3x105 or 2x105 cells per well, respectively, in flat-bottom, 96-well plates in IMDM supplemented with 10% FBS (Atlanta Biologicals), 1 mM Sodium Pyruvate (Lonza), 100 μM non-essential amino acids (Lonza), and 100 U/mL Pen/Strep (Lonza). For monocyte and tumor co-culture experiments, monocytes were cultured with fixed allogeneic tumor cells at a 3 to 1 ratio. Cells were subsequently incubated with immunostimulatory agents or controls for 18-36 hours at 37°C with 10% CO2. Cellular activation was measured through cell surface expression of activation markers and cytokine production. Surface marker expression levels were measured by flow cytometry with antibodies against CD40 (5C3, BD), CD86 (IT2.2, BD), HLA-DR (L243, BD), and CD16 (3G8, BD), CD14 (MΦP9, BD), and CD123 (7G3, BD). PBMCs were analyzed by flow cytometry with antibodies against the aforementioned activation markers as well as lineage markers CD19 (HIB19, BD), CD56 (B159, BD), CD3 (SK7, BD), CD4 (OKT4, BioLegend), CD8 (SK1, BD) and CD69 (FN50, BD). In all cases, cell culture supernatant was collected following 18 or 36-hour incubation, and cytokine production was measured using either cytokine bead array kits (Human Inflammatory Cytokine Kit, BD Biosciences) and ELISA (TNFα Human ELISA kit, eBiosciences). Mass Cytometry PBMCs were isolated from healthy donor blood and stimulated with ISAC, a mixture of antibody and adjuvant, or no stimulus for 5-15 minutes at 37°C. Following stimulation, cells were fixed by adding 16% PFA (Electron microscopy sciences) to a final concentration of 1.6% and incubated for 10 minutes at room temperature (RT). Fixed cells were washed with cell staining medium (CSM; PBS with 0.5% BSA and 0.02% sodium azide (all Sigma) and barcoded using a palladium-based approach as previously described 54. Barcoded samples were combined into one composite sample for surface staining.

Surface staining was performed by adding antibodies (Supplemental Table 2) in CSM and incubating for 30 minutes at RT. Anti-human antibodies were either purchased conjugated (Fluidigm) or conjugated to heavy metal isotopes using the MaxPar X8 antibody-labeling kit (Fluidigm) according to the manufacturer’s recommendations. Surface-stained cells were washed once in CSM and permeabilized with MeOH for 10 minutes on ice. Permeabilized cells were washed twice with CSM and antibodies against intracellular antigens were added for 30 minutes at RT. Cells were washed with CSM and finally resuspended in intercalation solution (1.6% PFA in PBS, 0.02% saponin (Sigma), and 0.5 μM iridium-intercalator (Fluidigm)) overnight at 4°C. Before data acquisition, samples were washed once in CSM and twice in ddH2O. All samples were filtered through a cell strainer (Falcon) and resuspended at 1x106 cells/mL in ddH2O supplemented with 1x EQ four-element calibration beads (Fluidigm) and data acquired on a CyTOF2 mass cytometer (Fluidigm). Signaling responses were evaluated using the SPADE implementation within Citibank 29. Analyses were run on all cells (no down-sampling), choosing the stated number of clusters and utilizing all relevant surface markers but no intracellular markers for clustering. Immune cell lineages were annotated based on their high-dimensional surface protein expression patterns. Statistical analyses were conducted in R, an open-source statistical software environment. Within each cell subset, treatment group, and donor, average fold change was computed for the following twenty phospho-protein markers: p-Src, p-STAT5, p-cMET, p-AKT, p-MAPKAPK2, p-SHP2, p-SLP76, p-IRF-7, p-ZAP70/SYK, p-CREB, p-NFKB, p-PI3K, IKB (total), p-PLCG1, p-ERK1/2, p-p38, p-BTK/ITK, p-S6, p-STAT3, and p-JNK/SAPK. The average fold change was weighted by the total number of cells in each cluster, as defined following SPADE clustering. Paired t-tests were conducted to estimate the difference in average fold change, and to test the null hypothesis that there was no difference in average fold change of each marker across treatment and control conditions.

Xenograft Tumor Model Experimental Procedure Tumor cell lines were purchased from ATCC (NCI-N87, HCC1954, and COLO205) or AddexBio (JIMT-1) and grown according to the manufacturer’s guidelines. Cells were harvested when they reached 80-90% confluency by detaching with Accutase (Stemcell), washed with PBS, resuspended at 40 x 106 cells/mL in PBS, and placed on ice for no longer than two hours. Immediately before implantation, suspended cells were mixed with an equal volume of Cultrex PathClear BME, Type 3 (R&D Systems), and 100 μL of the mixture (2 x 106 cells) were implanted subcutaneously into the right flank of 6-8-week-old female mice as follows: NCI-N87 and COLO205 cells were implanted in NSG mice (Jackson Laboratory), HCC1954 and JIMT-1 cells were implanted in Rag2/IL2rg double knockout (Taconic), and HCC1954 (for assessment with T785-ISAC) were implanted in NSG or SCID/beige mice (Jackson Laboratory or Taconic and Envigo). Tumor size was recorded twice a week and was estimated using the following formula: (length x width2)/2. Once tumors reached 50-100 mm3, usually within one week from tumor implantation, treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. Trastuzumab (EirGenix; EG12014), Trastuzumab ISAC, or Isotype ISAC was prepared in PBS at 1 mg/mL and administered at 5 mg/kg intraperitoneally every five days (Q5D) for a total of six doses. Mice whose tumors exceeded 2000 mm3 were euthanized.

HCC1954 and MMC Cellular Depletion Experimental Procedure To deplete macrophages and other phagocytic cells, mice bearing HCC1954 or MMC tumors were treated via intraperitoneal injection two days before the initial ISAC treatment with clodronate liposomes or control liposomes not containing clodronate as a negative control (Encapsula, Catalog # CLD-8901) and then treated twice a week for a total of three weeks. To deplete neutrophils, mice were treated with depleting antibodies 1A8 to deplete Ly6G+ neutrophils only (BioXCell Catalog # BE0075-1) or RB6-8C5 to deplete Gr1+  cells, which includes Ly6G+ neutrophils and Ly6C+ cells (BioXCell Catalog # BE0075) or isotype controls 2A3 and LTF-2 (BioXCell Catalog # BE0089 and BE0090, respectively) two days before the initial ISAC treatment, then treated twice a week for three weeks. Depletion of cells in the blood was confirmed by flow cytometry (Supplemental Figure 24). Tumor Cytokine & mRNA Analysis Procedure Tumors were implanted as previously described and were collected for analysis at the indicated time points post-treatment. For immunohistochemistry and mRNA analysis, tumors were split, and mRNA was analyzed with the NanoString mouse pan-cancer immune profiling panel. Data analysis was performed with nSolver Advanced Analysis Software according to the manufacturer’s recommendations. Volcano plots were drawn with the ggplot2 package in R. To obtain tumor lysates for cytokine analysis, tumor fragments were kept in a pre-made buffer (1 Pierce Protease Inhibitor Tablets (Thermo Fisher, Catalog #A32965) dissolved in 20 mL of T-PER Tissue Protein Extraction Reagent, Thermo Fisher, Catalog #78510) in GentleMACS M Tubes (Miltenyi, Catalog #130-093-236) immediately after necropsy. Tumor samples were then dissociated using a GentleMACS Octo Dissociator. All samples were spun down for 5 minutes at 300g after. Supernatants were then collected into microfuge tubes and were spun down again with a microcentrifuge. Cytokine analysis was performed on supernatants by MSD. Immunohistochemistry was performed using hematoxylin and eosin to identify tissue architecture, as well as for the following markers where indicated in studies: CD11c (CST Catalog #87585) and CD8 (CST Catalog #98941).

Figure 4: T785-ISACs elicit robust anti-tumor immunity in trastuzumab-resistant tumor xenograft models. (a-d) SCID/Beige or NSG mice were implanted with the HCC1954 tumor cell line and were randomized when the tumor volume reached 50 – 75 mm3. (a) Mice were treated once by intraperitoneal (IP) with 5 mg/kg trastuzumab T785-ISAC or trastuzumab, or with molar equivalent dosing with a mixture of trastuzumab and T785 to the ISAC via intratumoral (IT) injection. Data are from one experiment with n=3-5 mice per group. (b) Mice were treated via intraperitoneal injection with 5 mg/kg of trastuzumab, trastuzumab-ISAC, rituximab, or rituximab-ISAC Q5DX6 (n= 5 mice per group). (c) Mice were treated via intraperitoneal injection with 5 mg/kg of trastuzumab, trastuzumab T785-ISAC, trastuzumab N297A-ISAC, or trastuzumab TLRnull-ISAC (n=5 mice per group). (d) HCC1954 tumor cells were implanted into SCID/Beige mice, and cells of interest were depleted before and during trastuzumab T785-ISAC treatment (5 mg/kg, Q5DX3) using anti-Ly6G antibody (rat IgG2a control), clodronate liposomes (control liposomes as control) or anti-Gr1 antibody (rat IgG2b control). Data are from one experiment with n=4-6 mice per group. (e, f) NSG or Rag2/IL2rg double knockout mice were implanted with the JIMT-1 tumor cell line and randomized when the tumor volume reached 50 – 75 mm3. Mice were subsequently treated with the indicated treatments at 5 mg/kg intraperitoneally with a frequency of (e) Q5DX6 or (f) Q5DX5 (n=3-6 mice per group). (b-f) The data shown are representative of at least two experiments. (g-j) HCC-1954 tumors were harvested from cohorts of mice at the indicated time points after treatment with a single dose of 5 mg/kg ISAC or control antibodies. Tumors were processed for flow cytometry analysis, NanoString mRNA quantification, or formalin-fixed and paraffin-embedded for immunohistochemistry. (g) Volcano plots depict log2 fold change of gene expression in treated vs isotype control tumors measured by NanoString at 24 hours (n=5 mice per group). Changes with an adjusted p-value of < 0.05 are shown in red. (h) Gene signature scores quantified by nSolver Advanced Analysis Pathway Score for tumors analyzed 24 hours after treatment (n=5 mice per group). (i) Flow cytometry analysis of tumoral cellular composition 24 hours and 7 days following treatment (n=2-4 mice per group). Data are representative of at least 2 experiments. (j) Representative images as well as quantification of F4/80 and CD11c IHC of tumors harvested 9 days after each indicated treatment. Scale bars are 50 μm. (a-j) Data are shown as mean with SEM and statistics are shown with *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Syngeneic Tumor Model Experimental Procedure MMC cells were obtained from Dr. Disis (University of Washington) and were cultured in RPMI 1640 supplemented with 20% FBS, 1% penicillin-streptomycin, and L-glutamine at 37°C and 5% CO2 until 80-90% confluent. CT26 cells (ATCC) transfected to stably express rat HER2 protein were grown in RPMI 1640 supplemented with 10% FBS and 600 μg/mL G418 selective antibiotic (Thermo) at 37°C and 5% CO2 until 80-90% confluent. Once sufficiently confluent, cells were harvested by detaching with Accutase (Stemcell) and washing with PBS. Harvested cells were resuspended at 20 x 106 cells/mL (MMC) or 5 x 106 cells/mL (CT26-rHER2) in PBS and kept at 4°C/on ice until use. 100 μL of suspended cells were implanted subcutaneously into 6-8-week-old FVB/N-TgN(MMTV- Erbb2)NK1Mul/Jmice (MMC model, The Jackson Laboratory) or female BALB/c mice (CT26 model, The Jackson Laboratory). Tumor size was measured and recorded twice a week for the duration of the study. Tumor volume was estimated using the following formula: (length x width2)/2. Once tumors reached 200-500 mm3 (MMC) or 50-100 mm3 (CT26-rHER2), usually within one week from tumor implantation, treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. The anti-rHER2 mAb (BioXCell; 7.16.4) or anti-rHER2-ISAC were administered intraperitoneally (frequency as indicated in figure legends) for the duration of the study. Mice whose tumors exceeded 2000 mm3 were euthanized. T cell depletion was assessed in animals using CD4 (BioXCell; GK1.5) or CD8 (BioXCell; YTS 169.4) cell-depleting antibodies. Depletion was initiated before tumor implantation and continued with twice-weekly administration of 200 μg per antibody. For tumor rechallenge, CT26 and 4T1 cell lines were implanted at 5 x 106 cells per tumor line, and tumor naïve mice were challenged with both cell lines as controls.

Supplementary Material Refer to the Web version on PubMed Central for supplementary material. Acknowledgments: The authors wish to thank P. Basto, N. E. Reticker-Flynn, T. Prestwood, and B. Mallet for their helpful discussion. We also thank the Stanford Blood Center Flow Cytometry Core, specifically L. Tolentino, O. Choi, and N.Wu. We also extend our appreciation to Dr. Mary L. (Nora) Disis and Denise Cecil from the University of Washington who provided the MMC tumor cell line.

Funding: S. E. Ackerman was supported by a Stanford BioX Bowes Fellowship.

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Figure 6: CL264-containing ISACs elicit tumor clearance in HER2 medium expressing xenografts and T cell-mediated tumor clearance, immunologic memory, and epitope spreading in a syngeneic tumor model. (a) Chemical structures of T785 and CL264 adjuvants used for ISAC generation. (b) NSG mice implanted with the HCC1954 tumor cell line were randomized when the tumor volume reached 50 – 75 mm3. Mice were treated once via intraperitoneal injection of 5 mg/kg trastuzumab, trastuzumab T785-ISAC or trastuzumab CL264-ISAC. Tumors were harvested 20 hours post-administration, processed to a single cell suspension, and analyzed by flow cytometry to assess activation of tumor-infiltrating myeloid APCs. (c-d) NSG or Rag2/IL2rg double knockout mice were implanted with the indicated human tumor cell line and randomized when the tumor volume reached 50 – 75 mm3 (HCC1954) or 75 – 150 mm3 (JIMT-1). Mice were treated via intraperitoneal injection with 5 mg/kg of rituximab, trastuzumab, trastuzumab T785-ISAC, trastuzumab CL264-ISAC or the respective isotype-ISACs, every 5 days for a total of 6 treatments. (e) rHER2 expression was measured on CT26-rHER2 tumor cells in culture before implantation (left flow plot) and in tumors nine days post-implantation (right flow plot) by flow cytometry with fluorescently-conjugated anti-rHER2 antibody (red) or isotype control (blue). (f) Balb/c mice were implanted with the CT26-rHER2 tumor cell line and were randomized when the tumor volume reached 50 mm3. Mice were then treated via intraperitoneal injection with 10 mg/kg of mouse anti-rat HER2 or mouse anti-rat HER2 CL264-ISAC every 5 days for a total of 6 treatments. Data shown are from 1 experiment with 8 mice per arm and are representative of 3 experiments.

(g) Anti-rat HER2 CL264-ISAC treated mice that experienced complete tumor regression for >21 days after their last treatment were challenged with parental CT26 (left flank) and 4T1 cell lines (right flank), with or without CD4 and CD8 T cell depletion (n =3 each). Tumor naïve mice (n = 6) challenged with the parental CT26 and 4T1 cell lines were included as controls. (a-i) Data are shown from individual experiments with 3-6 mice per arm and are representative of 1-4 experiments with a minimum of 3 mice per arm in each experiment. Data are shown as mean with SEM and statistics are shown with *P<0.05,**P<0.01, ***P<0.001, and ****P<0.0001.

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