Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking LAIR-collagen interaction

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1). Leukocyte associated immunoglobulin-like receptor-2 (LAIR-2) is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 and that LAIR-2 could release LAIR-1 mediated immune suppression. Analysis of public datasets shows high LAIR-2 expression being associated with a favorable outcome in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 reduces tumor growth and increases T cell expansion and effector function in humanized tumor models. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a powerful new strategy for cancer immunotherapy for immune-excluded tumors.


Introduction 41
The introduction of immune checkpoint blockade therapies in the clinic has increased cancer treatment 42 options for a wide range of tumors leading to unprecedented and long-lasting clinical responses. However, 43 not all patients show the same degree of response and not all tumors respond to these therapies (1, 2). 44 Thus, identifying novel checkpoints and developing ideal combinations of immunotherapies is essential to 45 optimize and enhance the efficacy of treatment, achieving durable anti-cancer effects with reduced side 46 effects. 47 The extracellular matrix (ECM) is a major structural component in all tissues. It comprises a non-cellular 48 meshwork of proteins, glycoproteins, proteoglycans and polysaccharides with collagens as the most 49 abundant protein. Ongoing ECM remodeling ensures tissue integrity and function, with collagens being 50 synthesized and degraded in a highly regulated manner (3). At least 28 different collagens comprised of 51 at least 43 genes have been identified from which 4 have transmembrane domains allowing expression 52 on the cell surface (4). The ECM functions not only as a scaffold for tissue organization but also provides 53 critical biochemical and biomechanical cues that instruct cell growth, survival, differentiation and 54 migration and regulate vascular development and immune function (5). 55 Several epithelial cancers including breast, pancreatic, colorectal, ovarian, and lung cancer are 56 characterized by a dense ECM where high collagen content correlates with poor prognosis (6). Indeed, 57 ECM or "Matrisome" signatures associated with cancer type and stage of disease have been described (7-58 10). Cancer associated fibroblasts (CAFs) (11,12), macrophages and tumor cells themselves (7,13,14) all 59 contribute to increased collagen production during cancer progression. 60 The capacity of tumors to induce remodeling of collagens in the tumor microenvironment (TME) was 61 primarily thought to create a suitable microenvironment for tumor cell growth. We now consider 62 abnormal collagen production, composition and organization in the ECM-TME a cause of immune 63 human cancer types when compared to matched healthy tissues or non-tumoral adjacent healthy tissues 106 ( Figure 1A, middle graph). By stratifying patients for high and low LAIR-1 mRNA expression we observed 107 that patients with high LAIR-1 mRNA expression had lower survival probability in 4 out of 21 cancers, 108 namely LGG, ESCA, LUAD and LUSC ( Figure 1C). We also observed that LAIR-2 mRNA expression, despite 109 being at lower levels than collagen and LAIR-1, was significantly upregulated in 14 out 21 tumors 110 compared to healthy tissue ( Figure 1A bottom graph). We posited that increased mRNA expression of 111 LAIR-2, the soluble decoy for the LAIR-1 inhibitory receptor, may be associated with improved overall 112 survival. When patients were stratified by high and low LAIR-2 mRNA expression, high expression of LAIR-113 2 mRNA was indeed associated with increased overall survival probability in 6 out of 30 cancers analysed, 114 namely HNSC, STAD, thyroid carcinoma (THCA), thymoma (THYM), skin cutaneous melanoma (SKCM) and 115 breast invasive carcinoma (BRCA) ( Figure 1D). These data support the development of a therapeutic 116 intervention that can inhibit LAIR-1 checkpoint receptor interaction with collagen in the tumor 117 microenvironment of solid cancers. 118 119

NC410, a LAIR-2 human IgG1 fusion protein blocks collagen interaction with LAIR-1 120
Taking advantage of a natural decoy system that would both target cancers and reverse immune 121 inhibition, we generated a LAIR-2 human IgG1 Fc fusion protein for therapeutic use named NC410 ( Figure  122 2A). This fusion protein exists as a dimeric protein due to the cysteine bonding in the Fc portion of the 123 protein. NC410 binds to collagen I and III with high avidity by Octet based binding studies and is cross-124 reactive to multiple species including rat and mouse due to the conserved nature of collagens across 125 species ( Figure 2B). Human LAIR-2 binds with much higher affinity to collagens than human LAIR-1, as 126 reported elsewhere (42). NC410 completely blocks human LAIR-1 binding to collagen I, supporting the 127 high avidity interaction of NC410 and its role as a LAIR-1 decoy therapeutic ( Figure 2C). We also 128 determined whether NC410 prevents LAIR-1 mediated signaling ( Figure 2D, 2E). To do so, we used a 129 reporter cell line that expresses the human LAIR-1 extracellular domain (ECD) fused to CD3z, thus 130 conferring positive signaling capacity to LAIR-1 upon interaction with collagen ligands, and an  reporter to visualize LAIR-1 mediated signal induction (21). Using flow cytometry ( Figure 2D) and Incucyte 132 microscope imaging ( Figure 2E and Sup. Figure 1), we observed a dose-dependent inhibition of LAIR-1 133 NFAT-GFP reporter activity by NC410, indicating that NC410 inhibits collagen-mediated LAIR-1 signaling 134 in a dose-dependent fashion. 135

NC410 promotes T cell expansion and anti-tumor activity in humanized tumor models 137
To study the effect of NC410 on T cell function in vivo, we adoptively transferred human PBMCs into NSG 138 mice. In this model, human xeno-reactive T cells against mouse antigens expand and cause xenogeneic-139 graft versus-host disease (xeno-GVHD). This model is often used to assess if targeting T cell stimulatory or 140 inhibitory pathways alters xenogeneic T cell activity in vivo. We adapted this model by including the mouse 141 tumor cell line P815 ( Figure 3A). In this model, human PBMCs were injected intravenously on day 0, and 142 P815 cells were injected subcutaneously a day later. NC410 or control Fc protein was administered on 143 days 1 and 3 ( Figure 3A). NC410 promoted the expansion of human CD8 + T cells on day 13, but not on day 144 6 in this model ( Figure 3B). This was accompanied by reduced tumor growth of P815 after day 14 ( Figure  145 3C). 146 To determine if the T cell promoting and anti-tumor effects observed in the xenogeneic P815 model 147 correlated with the capacity of NC410 to elicit T cell anti-tumor activity against an allogeneic human 148 tumor, we developed a humanized subcutaneous tumor model using the HT-29 colorectal cancer cell line, 149 which expresses collagens (Sup. Figure 2). In this model, NSG mice are injected intravenously with human 150 PBMCs, and one day later subcutaneously with HT-29 cells ( Figure 3C). NC410 or control treatments began 151 on the same day as tumor implantation. NC410 treatment at 10mg/kg significantly reduced tumor growth 152 compared to isotype control ( Figure 3E). After titration of NC410, tumor volume was reduced in a dose 153 dependent manner ( Figure 3F). To investigate whether binding of NC410 to HT29 cells elicits antibody-154 dependent cellular cytotoxicity (ADCC), we performed in vitro cytotoxicity assays with HT-29 cells, using 155 human PBMC as source of effector cells. We confirmed that HT-29 single cells expressed collagen and that 156 NC410 was able to bind these cells after the EDTA treatment necessary to prepare the cells for the 157 chromium release assay (Sup. Figure 3). In a short-term in vitro chromium release cytotoxicity assay, no 158 differences in HT-29 killing by PBMC were observed between NC410, isotype treated samples and medium 159 ( Figure 3G) suggesting that enhancement of ADCC by NC410 is not contributing to tumor reduction in vivo. 160 Thus, we established that NC410 has the capacity to induce T cell expansion and reduction of tumor 161 growth in different in vivo tumor models. 162

NC410 enhances anti-tumor T cell responses 164
Cytokines and chemokines mediate the host-response to cancer by activating and directing the trafficking 165 of immune cells into the TME (43, 44). To determine if NC410 promoted infiltration and localized activity 166 of T cells in the TME, immune profiling of HT-29 tumors and spleen tissues was performed at day 27 after 167 treatment (10 mg/kg dose). HT-29 tumors were removed from euthanized mice, weighed, and dissociated 168 for analysis of T cells and soluble factors. For equal comparison of systemic response, the spleen was 169 harvested by identical means for analysis. Analysis of T cell numbers within the tumor showed a significant 170 increase in the number of CD4 + and a trend of increased CD8 + T cells in tumors treated with NC410 171 compared to isotype control ( Figure 4A and B). To determine the effector capacity of tumor infiltrating T 172 cells (TILs) from NC410-treated mice, we re-stimulated TILs from digested tumors with anti-CD3+anti-173 CD28 for 5 hours and performed intracellular staining to examine IFN-γ and TNF-α production. After re-174 stimulation a significant increase in IFN-γ + and IFN-γ + TNF-α + double positive cells was observed in the 175 NC410 treatment group compared to control. ( Figure 4C). Based on this observation, we further assessed 176 cytokines and chemokines in the local TME vs peripheral spleen on day 27 ( Figure 4D-E and Sup figure 5). 177 Analysis of cytokines indicated that IFN-γ and granzyme B were significantly increased in the TME, but not 178 soluble CD40L ( Figure 4D). Increased expression of CD40L and granzyme B was observed in the spleen 179 ( Figure 4D). Chemokine analysis indicated that CXCL10, 11 and 12 were all significantly increased in the 180 TME, and significantly decreased in the spleen ( Figure 4E). Importantly, the concentration of all three 181 chemokines correlated with tumor reduction (Sup. Figure 4). These results support a role for NC410 in 182 enhancing the recruitment of T cells and liberating their effector function in the TME. 183

NC410 promotes collagen remodeling 185
Remodeling of ECM is pivotal to the development and progression of cancer. Recently it has been 186 proposed that specific collagen-derived biomarkers reflecting the turnover of collagens may be used as 187 tools to non-invasively interrogate cell reactivity in the TME and predict response to treatment (45-47). 188 Because NC410 both engages collagens and promotes local T cell and immune responses, it was surmised 189 that NC410 treatment may result in collagen remodeling. Therefore, we determined whether NC410 190 treatment increased collagen degradation products (CDPs) in the serum of HT-29 tumor bearing mice 191 injected with PBMC over the course of tumor growth and rejection mediated by NC410 ( Figure 5A and B). 192 Nine CDPs were analyzed prior to experiment initiation and during four weeks of tumor growth (Figure 193 5C). While there was no change in most CDP, it was interesting that the concentration of two CDPs 194 significantly increased in the serum of NC410 treated mice in comparison to controls. C6M, a collagen VI 195 MMP-2 CDP, and C4GzB, a collagen IV Granzyme B CDP were significantly increased at week 4 ( Figure 5D) 196 in the NC410 treated group in comparison to the control group. Interestingly, this increase in serum CDPs 197 was observed at the time of NC410 mediated tumor eradication suggesting that the CDPs were derived 198 from the tumor and that the increase in CDPs was a result of T cells activation and effector function. 199 200 NC410 binds collagen rich tumors with an immune excluded phenotype 201 In order to identify tumor types and/or patient groups that would benefit from this therapy, we performed 202 immunohistochemical analysis of serial tissue sections from seven different tumor types, of which we 203 analyzed biopsies from ten patients per tumor type. Sections of head and neck squamous cell carcinoma 204 (HNSC), glioblastoma (GBM), melanoma, non-small-cell lung carcinoma (NSCLC), high-grade serous 205 carcinoma (HGSC), pancreatic ductal adenocarcinoma (PDAC), and stomach adenocarcinoma (STAD), 206 were stained for hematoxylin and eosin, Masson's Trichome, anti-LAIR-1, NC410 and the immune cell 207 markers, CD45, CD3, CD68 and CD163 ( Figure 6A). NC410 binding co-localized with Masson's Trichrome 208 positive collagen areas within the TME and LAIR-1 + immune cells were present in all tumors ( Figure 6B, 209 Sup. Figure 5, heathy tissue Sup. Figure 6). Both myeloid and lymphoid cells within the TME expressed 210 LAIR-1 (Sup. Figure 7). NC410 binding was highest in pancreatic cancer in agreement with a collagen-rich 211 microenvironment ( Figure 6C). Head and neck squamous cell carcinoma had the highest number of LAIR-212 1 + cells ( Figure 6C). Importantly, LAIR-1 + cells were enriched in NC410 positive areas ( Figure 6D) suggesting 213 that LAIR-1 + cells were entrapped in the collagen matrix. Most human solid tumours exhibit distinct 214 immunological phenotypes being divided into immune inflamed, immune excluded, and immune desert 215 tumors on the basis of immune cell infiltrate and localization (48, 49). By characterizing our cohort 216 according to these immunological phenotypes ( Figure 6E, Sup. Figure 8), we observed that NC410 binding 217 was particularly increased in immune excluded tumors, namely HNSC, melanoma, NSCLC and STAD. 218 ( Figure 6F). Thus, in immune excluded tumors, LAIR-1 + cells are sequestered in collagen-rich areas where 219 NC410 can bind. 220

Discussion 221
Cancer cells have evolved multiple mechanisms to escape immune surveillance, such as defects in antigen 222 presentation machinery, the recruitment of immunosuppressive cell populations and the upregulation of 223 negative regulatory pathways, all resulting in hampered effector function of immune cells and the 224 abrogation of antitumor immune responses (50). Tumor progression is also accompanied by extensive 225 remodeling of the extracellular matrix leading to the formation of a tumor-specific ECM, which is often 226 more collagen-rich and of increased stiffness (51, 52). Collagen expression and density have been shown 227 to be associated with a worse prognosis, either by directly promoting tumor growth or by preventing 228 immune cell infiltration (8). 229 We have shown that a combined collagen signature was associated with a worse prognosis in 13 out of 230 21 tumor types supporting the notion that increased collagen expression is detrimental for overall survival 231 in tumors. Importantly, LAIR-1 mRNA expression was associated with a worse prognosis in 4 out of 21 232 tumors and LAIR-2 mRNA expression was associated with a better prognosis in 6 out of 21 tumors. This 233 indicates that targeting LAIR-1-collagen interaction might be helpful in a specific group of tumors. 234 It has been demonstrated that collagens can also directly modulate T cell function(53-55), for instance 235 through the inhibitory collagen receptor 27). We hypothesize that increased expression and 236 remodeling of collagen in the TME serves to set a threshold of T cell activation through LAIR-1 and is 237 employed by tumor cells to escape immune surveillance. In this study we show that disrupting collagen 238 interaction with LAIR-1 by means of a dimeric LAIR-2 Fc fusion protein has a therapeutic effect in cancer 239 models. NC410 administration in humanized mouse tumor models results in a reduction of tumor volume 240 by increasing T cell expansion and cytotoxic activity but does not enhance ADCC in vitro. NC410 binds to 241 collagen-rich areas of tumors, where LAIR-1 + cells are sequestered. NC410 also binds to healthy collagen, 242 therefore next to the direct effect on T cell mobilization and activation in the tumor, systemic effects could 243 also add to its therapeutic potential. 244 During ECM turnover, proteolytically cleaved matrix degradation fragments are released into the systemic 245 circulation (3). These small protein fragments containing specific protease-generated neo-epitopes, or 246 'protein fingerprints', can be used as serological biomarkers directly reflecting disease pathogenesis. 247 Several studies have shown that serum levels of collagen degradation fragments are elevated in cancer 248 patients compared to healthy controls (56-58), and that checkpoint blockade may alter serum CDP levels 249 (47). We observed that specific fragments generated by degradation of collagen VI by MMP-2 (C6M) and 250 collagen IV by granzyme B (C4GzB) were increased after NC410 treatment. The increase of circulating C6M 251 and C4GzB coincided with reduction of tumor volume suggesting that these fragments might be generated 252 by increased infiltration and/or activation of T cells in the TME. C6M and C4GzB collagen fragments have 253 the potential to be biomarkers of NC410 response, which will be investigated in a recently initiated NC410 254 first-in-human clinical trial (NCT04408599). 255 In the era of personalized medicine, it is very important to address how to select patients that are likely 256 to benefit from immune checkpoint blockade therapy. Within different tumor types a distinction based 257 on the presence and location of immune infiltrates can be made, separating patients into having immune 258 excluded, immune inflamed and immune desert tumors. Currently, immune inflamed tumors, where the 259 majority of immune cells are present throughout the neoplastic cells, have been associated with better 260 response to currently available checkpoint blockade therapy compared with the other phenotypes (59, 261 60). The immune-excluded phenotype is characterized by the presence of immune cells that cannot 262 penetrate the parenchyma of the tumors but instead are located in the stroma that surrounds the cancer 263 cells (61). These tumors are generally resistant to current checkpoint blockade therapy (62). We 264 performed an immunohistochemical screen across 7 tumor types, analyzing biopsies of 10 individual 265 patients per tumor type, and observed that immune excluded tumors had the highest NC410 binding. 266 Importantly we observed that LAIR-1 + cells were present within these NC410-binding collagen rich areas. 267 This might indicate that an abundance of collagen keeps the immune cells trapped in the tumor stroma, 268 possibly by binding to LAIR-1. Treatment with NC410 could block this interaction resulting in LAIR-1 + cell 269 activation and infiltration into the tumor nests, promoting tumor clearance. 270 LAIR-2 acts as a decoy receptor by binding to collagen with higher affinity than LAIR-1 and therefore 271 antagonizes LAIR-1 inhibitory function (33, 34). Circulating LAIR-2 protein concentration is low or 272 undetectable in healthy individuals (33,42). In the presence of limited endogenous LAIR-2, LAIR-1 can 273 bind collagens, thereby allowing the inhibitory receptor to signal and prevent or reduce T cell activity 274 and/or retain LAIR-1+ cells in collagen-rich areas. In the TME increased levels of collagens, in absence of 275 increased LAIR-2, will therefore promote tumor immune escape. Our analysis of TCGA data indeed 276 revealed that enhanced expression of endogenous LAIR-2 in some cancers associated with better 277 prognosis, suggesting that further increasing LAIR-2 in vivo would have a therapeutic advantage. NC410 278 has a higher avidity to collagen than endogenous LAIR-2 due to its dimeric structure, since in vivo LAIR-2 279 is expressed as a monomer, enhancing its potential to block the inhibitory capacity of membrane-bound 280 LAIR-1. NC410 binds both healthy and tumoral collagen and theoretically could be sequestered before 281 reaching the tumor site. We hypothesize that the avidity of NC410 towards tumoral collagen is higher 282 than to healthy collagen, therefore resulting in a therapeutic effect, but this needs additional study. Other 283 LAIR-1 ligands, such as C1q have been shown to be increased in tumors (19) and may also provide a local 284 inhibitory effect that could be potentially reversed by NC410. 285 Taken together, tumor patients with collagen rich tumors that present an immune excluded phenotype 286 and low endogenous LAIR-2 expression would be predicted to benefit the most from NC410 therapy. Our 287 immunohistochemical analysis points to HNSC, melanoma, NSCLC and STAD as immune excluded tumor 288 types with the highest NC410 binding. In agreement with our hypothesis that blocking LAIR-1-collagen 289 interaction in these tumors would be beneficial, TCGA analysis showed that increased LAIR-2 mRNA at the 290 tumor site also increases survival probability in HNSC, melanoma, and STAD. Together, this may be 291 indicative that patients suffering from these particular tumors might benefit the most from NC410 292 treatment. 293 While conducting our study, an unrelated research group generated a structurally similar LAIR-2 Fc fusion 294 protein for use as an immune therapy for cancer to block LAIR-1 inhibitory function (63). Xu et al. showed 295 that LAIR-2 Fc reversed T cell inhibition in vitro and in vivo and promoted anti-tumor immunity in vivo. Our 296 models are non-overlapping with the models described in that study, independently supporting the 297 concept of LAIR-2 Fc for cancer immunotherapy. In addition, we now provide an immunohistochemical 298 rationale for LAIR-2 Fc treatment in certain tumor types by extensive analysis of human tumor samples, 299 as well as potential biomarkers for patient selection and response to therapy. 300 Our data support NC410 as a novel immunomedicine for targeting immune excluded collagen-rich tumors The human LAIR-1 and LAIR-2 genes were synthesized by GeneArt and genetically fused with the N 353 terminus of IgG1 Fc domain. A stable CHO cell line expressing recombinant human LAIR-1 Fc or LAIR-2 Fc 354 fusion protein was developed using the Lonza GS system. Briefly, 5 × 10 7 CHO cells were transfected by 355 electroporation using 80 μg of linearized plasmid DNA in a 0.4 cm cuvette. Following electroporation (300 356 V, 900 μF) cells were resuspended in 100 mL glutamine-free CD CHO medium (ThermoFisher). The 357 following day, MSX (Millipore) was added to a final concentration of 50 μM, and cells were monitored for 358 the next two weeks as prototrophic cells began to grow. Single clone of stably transfected cells was 359 cultured, and supernatant was harvested and purified by affinity chromatography. The protein purity was 360 determined by HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For the LAIR-1-361 collagen binding assay, LAIR-1 Fc was biotinylated with EZ-Link NHS-PEG4-Biotin (ThermoFisher), No-362 Weight Format (ThermoFisher) and free biotin was removed by ZebaSpin Desalting Columns 363 (ThermoFisher) following manufacturer's instructions. 364 365

Human PBMC preparation for in vitro experiments 366
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood of healthy donors (in agreement 367 with ethical committee of the University Medical Center Utrecht (UMCU) and after written informed 368 consent from the subjects in accordance with the Declaration of Helsinki) using standard Ficoll density 369 gradient centrifugation. Briefly, blood was diluted 1:1 with PBS and layered on top of 15 mL of Ficoll-Paque 370 (GE healthcare) in 50 mL conical tube. Suspension was centrifuged at 400 × g for 20 min at 20 °C in a 371 swinging bucket rotor without brake. The mononuclear cell layer at the interphase was carefully collected 372 and transferred to a new 50 mL conical tube. The cells were washed with PBS and centrifuged at 300 × g 373 for 10 min at 20 °C. The supernatant was discarded, and the cell pellet was washed twice with 50 mL PBS. 374 The isolated PBMCs were immediately used for in vitro studies. 375 376

Binding and Blocking Studies 377
Octet avidity analysis 378 A ForteBio Octet RED96 instrument was used for avidity assessments. The anti-human Fc antibody capture 379 sensors (ForteBio) were first loaded with LAIR-2-Fc followed by an association step where the loaded 380 sensor was dipped into wells containing human, mouse or rat collagen I (human, R&D Systems; mouse, 381 Ray Biotech; rat, Yo Protein) or collagen III (human, R&D Systems; mouse, Abbexa; rat, Yo Protein). NC410 382 protein was diluted in assay buffer (ForteBio) at 20 µg/mL and the collagen concentration ranged from 383 1.56 µg/mL to 100 µg/mL for collagen I and from 0.78 µg/mL to 50 µg/mL for collagen III. Data processing 384 was conducted using the Octet's Data Analysis 9.0 software. 385 386 Time Resolved Fluorometry (TRF) Immunoassay 387 EIA plates were coated with human collagen I (StemCell) in 0.01 N HCL (100 μL/well) overnight at 4 °C. 388 The following day, plates were equilibrated to ambient temperature and washed 3 times (300 μL/well) 389 with DELFIA Wash buffer (PerkinElmer). The plates were blocked for non-specific binding with 3% BSA 390 (200 μL/well, Millipore) for 1 hour. Plates were washed 3 times (300 μL/well) with DELFIA wash buffer and 391 an NC410-biotin and human LAIR-1 Fc mixture (50 μL/well) was added to plates and incubated for 2 hours 392 at ambient temperature. The plates were washed 3 times (300 μL/well) with DELFIA wash buffer. 393 Europium-labeled Streptavidin (Eu-SA) (100 μL/well, PerkinElmer) was diluted 1:1000 in DELFIA assay 394 buffer and was added to plates and incubated for 1 hour at ambient temperature. (ThermoFisher). Human CD4 + and CD8 + T cells were gated based on live/hCD45 + mCD45 -hCD3 + hCD4 + hCD8 -454 and live/hCD45 + mCD45 -hCD3 + hCD4 -hCD8 + respectively. 455 To prepare mouse blood T cells for staining, 80 -200 μL blood was treated with 3 mL ACK lysis buffer (KD 456 medical) to lyse the red blood cells for 5 min at RT followed by washing with PBS. The initial volume of 457 blood was recorded for calculation of cell counts per mL of blood according to the formula: Cell counts 458 per mL of blood= [acquired counts × 150 × 1000] ÷ [initial blood volume(μL) × 80]. 459 To prepare the single cell population from tumors for staining, tumor tissues were weighed, cut into small 460 pieces, and digested with mouse tumor dissociation kit (Miltenyi) and dissociated with gentleMACS 461 Dissociator (Miltenyi). The tumor weight was recorded for the normalization of cell counts. 462 To assess cytokines from tissues, tumor and spleen tissues were weighed and cut into small pieces in 463 1.5mL Eppendorf tube on ice. 200 μL of RIPA Lysis buffer (ThermoFisher) was added with proteinase 464 inhibitor (Roche) and 250 U/mL of DNase (Millipore) and the tissues were dissociated with pellet pestles 465 (Sigma) on ice. The samples were kept on ice for 30 min, vortexing occasionally. Centrifuge at 10,000 ×g 466 for 20 mins at 4 °C to pellet cell debris and then transfer the supernatant to a fresh Eppendorf tube without 467 disturbing the pellet. The tissue weight was recorded for the normalization analysis of cytokines. Scoring of the immune phenotype was based on the presence and distribution of CD3 positive 561 lymphocytes(61). Tumors with CD3 + cells in the tumor fields were scored "inflamed"; tumors with CD3 + 562 cells in their stroma, but without or with a relatively low amount of CD3 + in the tumor fields were scored 563 "immune excluded"; tumors with a lack of CD3 + cells in their stroma as well as in their tumor fields were 564 scored "immune desert". 565 For quantification of the immune cell counts, regions of interest (ROIs) were annotated on the tissue slides 566 by drawing circles with a diameter of 600 µm at five random spots within the NC410 binding part of the 567 tumor. Positive cells were quantified using the Positive Cell Detection tool. For each staining, stain vectors 568 and DAB cutoffs were determined based on a representative slide; the settings were kept the same for all 569 slides. Tumor area within the circles that was lost during staining procedure or that was negative for 570 NC410 binding was excluded from the analysis. 571 The percentage of NC410 binding to tissue within a tumor was calculated by dividing the stained area by 572 the total tumor area. The immune cell counts for each tumor were calculated by dividing the total number 573 of positive cells within the five ROIs by the total surface in mm 2 of these ROIs.   *** * *** *** ** ** *** * *** *** *** *** ** n.s. n.s. * * *** *** *** n.s. *** n.s. *** *** *** ** *** * *** *** ** *** *** ** n.s. n.s. n.s. *** * * * *** n.s. n.s. *** *** n.s. *** n.s. *** * *** ** * n.s. n.s. * ** *** n.s. n.s.