AACR 2023 XDC合集（一）

由于字数过多,分两篇发表,合集（一)摘要编号范围为383-3942,所有编号无英文字母。合集（二)摘要编号范围为3995-LB246，所有涉及英文字母的编号都在此。

AACR 2023会议关于XDC的报告数量大约有195篇，涉及ADC、PDC、RDC、iADC、SMDC、VDC和纳米粒等。

其中ADC包括iADC共有150篇，中国企业占有43篇，占比为28.6%，出场的中国企业分别为百奥赛图、恒瑞医药、启德医药、映恩生物、百利药业、宜联生物、康源久远、普方生物、百力司康、博锐生物、集萃药康、药明康德、礼新医药、普众发现/昂阔医药、荣昌生物、石药集团等。

从数量上来看，共有14家企业的报告篇数≥3篇，其中中国企业有6家。下图可以看到，全球ADC知名的国外企业基本都在图中了，国内企业则为ADC领域的新锐选手。     

图1 ADC报告篇数数量排名情况，≥3篇（丰硕创投整理）

下图则为本次会议的195篇报告情况，粉红色的为披露临床数据的报告。数据来源一般为本次报告摘要、公司官网和文献资料，网上没有链接来源的数据没有摆放进图里。

在这里推荐两篇报告，一篇是Zymeworks发表的，摘要编号为1538，讨论了linker稳定性与临床效果的关系，指出临床前Linker稳定性的数据并不能很好地反映临床效果，拿enhertu为例，人体的Payload AUC/总抗AUC数值除以猴子的Payload AUC/总抗AUC数值，等于26，细思极恐。

另一篇是Mythic发表的，摘要编号为5000，关于pH依赖性的内吞抗体设计，之所以对pH与内吞之间的关系感兴趣，是由于之前的昂科免疫CTLA-4的2亿美元首付款的交易事件，这可能是ADC抗体部分，除去解离常数、Fc段非特异性介导的毒性之外的新突破方向。

下面为RDC、SMDC和VDC。

下面为纳米粒。

图2 AACR 2023 XDC领域报告情况（丰硕创投整理）

Abstract 383: Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors

Activating ERBB2 (HER2) mutations occur across multiple malignancies, and although mutational hotpots vary by disease, HER2 mutations most frequently occur in the tyrosine kinase domain. Among lung cancer patients, the most common HER2 mutation is the exon 20 insertion mutations, Y772dupYVMA, which accounts for over 30% of all HER2 mutations in lung cancer. Recent clinical testing of trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), against HER2 mutant non-small cell lung cancer (NSCLC) demonstrated a confirmed objective response of 55%, a median duration of response of 9.3 months, and a median overall survival of 17.8 months, which ultimately led to the recent approval of T-DXd for HER2 mutant NSCLC. Acquired resistance to targeted agents remains a major clinical challenge, and HER2-mutant patients that initially respond to T-DXd will eventually develop progressive disease. Thus, understanding mechanism of T-DXd resistance and identifying targeted agents that remain effective in cells with acquired T-DXd resistance is of great clinical importance. We generated Ba/F3 cells expressing the HER2 YVMA mutation and assessed sensitivity to HER2 targeting agents by Cell Titer Glo assay. HER2 YVMA cells were sensitive to the EGFR/HER2 tyrosine kinase inhibitor (TKI) poziotinib and the HER2 ADC T-DXd. To generate HER2 ADC resistant cells, we cultured Ba/F3 HER2 YVMA cells in T-DXd continuously for 8 weeks at which point cells resumed rapid proliferation. Cell Titer Glo assay confirmed that Ba/F3 HER2 YVMA cells were sensitive to T-DXd with an IC50 of 195 μg/ml, whereas Ba/F3 HER2 YVMA T-DXd-resistant cells had an IC50 of greater than 1000 μg/ml. Notably, T-DXd resistant cells remained highly sensitive to the HER2 TKI poziotinib with an IC50 of 14 nM and 7.5 nM for T-DXd-resistant and parental cells, respectively. Moreover, T-DXd-resistant cells demonstrated reduced sensitivity to the topoisomerase inhibitor topotecan with an IC50 of 200 nM whereas parental cells had an IC50 of 66 nM, suggesting that acquired T-DXd resistance may be mediated by loss of sensitivity to the ADC payload. Collectively, these data demonstrate that HER2 TKIs such as poziotinib may retain anti-tumor cell activity in HER2 mutant tumor cells with acquired resistance to HER2 ADCs.

Abstract 438: Antibody drug conjugate using a novel anti-CDCP1 antibody with low hematopoietic stem-cell binding and rapid internalization showed high efficacy for solid cancer

CUB domain containing protein 1 (CDCP1) is a single transmembrane protein that is highly expressed in a broad range of solid cancers, including lung, breast, colorectal, pancreas, prostate, and ovarian cancers. Although no soluble ligand for CDCP1 is known, CDCP1 transduces signals resulting in cancer cell motility, epithelial mesenchymal transition, and metastasis by activating downstream signaling via homodimerization or heterodimerization with other cell surface molecules such as EGFR, HER2, and ITGB1. Owing to its important roles in cancer, CDCP1 has been reported as a promising therapeutic target for the treatment of solid cancers. While it is overexpressed in many of the abovementioned solid cancer types, several normal tissues such as esophagus, skin, and bone marrow hematopoietic stem cells (HSCs) also express CDCP1. To minimize the risk of toxicity, we generated novel anti-CDCP1 antibodies that bind weakly to bone marrow HSCs. Three isolated anti-CDCP1 monoclonal antibodies and their humanized versions showed weak binding to HSCs compared to other anti-CDCP1 antibodies reported from other groups. Although they only showed moderate binding activity to cancer cell lines, rapid internalization and efficient lysosomal trafficking was observed following CDCP1 binding. Subsequently, we generated antibody drug conjugates (ADCs) of the 3 anti-CDCP1 antibodies using PBD, MMAE, and MMAF as a cytotoxic payload with both a cleavable and non-cleavable linker. All constructed ADCs showed strong anti-tumor activity in multiple cancer cell lines in vitro. The cytotoxic activities of these ADCs were correlated to CDCP1 surface expression levels and internalization activity. The anti-CDCP1 ADCs showed strong anti-tumor efficacy in multiple cell line xenograft models. We selected the clone h14A043 for further evaluation owing to its observed potent anti-tumor activity both in vitro and in vivo. We then constructed h14A043-ATAC (Antibody Targeted Amanitin Conjugate), which has a site-specifically conjugated amanitin derivative with DAR 2 and an Fc domain mutation that reduces the effector function of human IgG1. h14A043-ATAC showed good anti-tumor potency both in vitro and in vivo. In human prostate cancer PC3 cell line xenograft model, we observed significant tumor growth delay following treatment by h14A043-ATAC with a single administration of 0.1 mg/kg. Furthermore, we observed complete tumor remission in 3 out of 8 mice at 40 days after a single administration of 1 mg/kg. In the non-GLP single dose toxicity study using cynomolgus monkeys, h14A043-ATAC showed good serum stability and no hematological toxicity. Our results suggest that ADC using an anti-CDCP1 mAb with weak binding to bone marrow HSC and sufficient internalization activity could provide a new treatment option for refractory solid cancer patients.

Abstract 455: Development of an antibody-drug conjugate panel targeting high grade serous ovarian cancer

Antibody drug conjugate (ADC) is a novel class of therapeutic agent which recently showed great success in both solid tumour and blood cancer. ADCs deliver potent cytotoxic payloads to tumour via cancer specific monoclonal antibodies. A few ADCs are currently evaluated in clinical trials for high grade serous ovarian cancer (HGSOC). However, most of them target FOLR1-alpha, a conventional HGSOC target which also expresses in vital organs including lungs. Thus, it is desirable to discover more specific antigen targets, and these new targets will be the foundation for the next-generation ADC for HGSOC.We propose that ovarian tumour specificity of an ADC target could contributed by three factors: 1. Lineage (e.g. fallopian tube marker FOLR1); 2. DNA amplification (e.g. ERBB2); 3. Other carcinogenesis process including epigenetic reprogramming. Then, by combining the GTEX, TCGA dataset with our own WES, long-reads RNA-sequencing and Mass spectrometry-based proteomics cohort, an array of differentially expressed genes in HGSOC tumours are identified. Out of over 50 candidates, two most promising targets are chosen for monoclonal antibodies (mAbs) production and subsequent ADC development. First, the affinity and specificity of the mAbs were confirmed by ELISA, flow cytometry and immunofluorescence. The cellular internalisation characteristics of these antibodies are confirmed by a fluorescence-based assay. Then, ADCs for these two targets are produced by conjugating monomethyl auristatin E payload to the mAbs via a cleavable linker. Finally, both ADCs show highly target-specific and sub-nanomolar cytotoxicity in model ovarian cancer cell lines. In conclusion, we identify a panel of HGSOC-specific ADC targets and develop two ADCs candidates for further pre-clinical and clinical investigations.

Abstract 456: Raptamer-drug conjugates as molecularly targeted cancer therapeutics

While antibody-drug conjugates (ADC) are an attractive modality for targeted cancer therapy, they face limitations. These include a reduced volume of distribution with limited penetration into solid tumors, and an expensive, long and complicated production process, especially for large-scale manufacturing. These factors hinder the activity of these medications in certain tumors, and limit equitable access to all population segments. Use of aptamers as a therapeutic targeting moiety, holds the promise of building on the success of ADCs. Aptamers have several advantages over mAbs as a targeting moiety including: (i) smaller size that enables better tissue/tumor penetration; (ii) chemical synthesis which allows rapid and affordable scale-up; (iii) superior safety profile from lack of immunogenicity and short half-life which reduces off-target adverse events; and (iv) room temperature stability. Therefore, the use of aptamers in conjunction with drugs (similar to ADCs) holds promise for tumor management. We have recently identified a novel therapeutic target for certain cancers (COF-01) using analysis of proteomics data and immunohistochemistry from patient-derived tumors. COF-01 was shown to have highly specific increases in protein and mRNA expression in several cancers including non-small cell lung cancer, head and neck cancer and pancreatic cancer. COF-01 expression correlated with poor overall survival. Here, we present a new therapeutic approach that targets COF-01-positive cells using next generation drug conjugated aptamers. We have generated novel next-generation base-modified ssDNA aptamers (Raptamers) against COF-01 using a cell- and protein-based selection that utilizes our proprietary bead-based oligonucleotide libraries. The binding affinities of these COF-01 Raptamer candidates were determined by concentration curves using biolayer interferometry. Dissociation constants for the COF-01 Raptamers ranged from 80 nM - 200 nM. Anti-COF-01 Raptamer-drug conjugates (Rap-DCs) were developed by conjugating the Raptamers to monomethyl auristatin F via a cleavable linker. In vitro efficacy data has shown that the COF-01 Rap-DCs can selectively induce cytotoxicity in COF-01-positive human cells. There was a significant decrease in cell viability of COF-01-positive cells when compared to COF-01-negative control cells. This study provides proof-of concept for the use of high-affinity Raptamers against novel molecular targets, such as COF-01. This platform can be used to develop Raptamers against emergent neo-antigens on cancer cells that have escaped other therapies.

Abstract 484: VIP236: A small molecule drug conjugate with an optimized camptothecin payload has significant activity in patient-derived and metastatic cancer models

Introduction: We previously presented the design and preliminary characterization of VIP236—an SMDC consisting of an αvβ3 integrin binder linked to an optimized camptothecin topoisomerase I (TOP1) inhibitor payload released by neutrophil elastase in the tumor microenvironment (Lerchen et al, Cancers 2022). Herein we evaluate VIP236 in patient-derived and metastatic models of cancer.

Methods: Subcutaneous patient-derived xenograft mouse models in non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), colorectal cancer (CRC), and triple negative breast cancer (TNBC) were treated with VIP236. Metastasis formation was measured by human DNA PCR in an orthotopic TNBC mouse model. Gastric cancer models included patient-derived and cell line xenograft mouse models. Tumor/control (T/C) ratios were calculated, and body weight was measured. Pharmacodynamic (PD) effect of VIP236 in a gastric cancer SNU16 xenograft mouse model was investigated. Tumor samples from vehicle- and VIP236-treated groups collected up to 144 hours postdose were analyzed for % positive γH2AX cells by a fit-for-purpose qualified immunohistochemistry assay.

Results: VIP236 is efficacious in vivo patient-derived models of NSCLC, RCC, CRC and TNBC with T/C ratios of 0.0, 8.9,14.5 and 5.6 compared with vehicle control (p<0.0001, CRC p<0.01). VIP236 showed statistically improved in vivo efficacy in gastric cancer models versus trastuzumab deruxtecan (a commercial antibody-drug conjugate [ADC] with a camptothecin payload) in NCIH87 (HER2high, p<0.05), SNU16 (HER2neg, p<0.001), and GXA3040 (patient-derived, HER2low, p<0.001) xenografts. VIP236 induced statistically significant tumor growth inhibition in a patient-derived CRC liver metastasis model (p<0.01) and statistically significant reduction in lung (p<0.001) and brain (p<0.01) metastasis from an orthotopic TNBC model was observed. Across all in vivo models no clinically significant weight loss or treatment-related mortality were observed. Furthermore, a time-dependent increase of γH2AX % positive cells in SNU16 in vivo VIP236-treated tumors confirms that VIP236 drives robust TOP1 inhibition and subsequent DNA damage.

Conclusions: In summary, VIP236 monotherapy efficacy in NSCLC, gastric, TNBC, RCC, CRC, and metastatic TNBC in vivo cancer models provides a rationale for clinical investigation in advanced metastatic solid tumors. In gastric cancer, VIP236 showed improved efficacy compared with the approved ADC, trastuzumab deruxtecan, regardless of HER2 expression. These results warrant further evaluation in clinical trials.

Abstract 540: c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery

Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of the c-MET antibody ABT-700 and the microtubule inhibitor monomethyl auristatin E (MMAE). Patients with EGFR wildtype (WT) nonsquamous non-small cell lung cancer with high c-MET expression had an overall response rate of 54% with Teliso-V. In addition to overexpression and amplification, genetic alterations in c-MET, such as exon 14 deletion (ex14del) and missense mutations in the tyrosine kinase domain (TKD), contribute to its constitutive activation. The efficacy of Teliso-V in context of these genomic backgrounds is currently unknown.

Methods: Recombinant retroviruses were used to express WT-c-MET, c-MET-TKD (D1228H/V/N and Y1230C/H mutations), c-MET-Y1003F/N (CBL E3 ubiquitin ligase binding site mutation), and c-MET-ex14del (47 aa juxtamembrane deletion) mutants in NIH3T3 and Ba/F3 cells. c-MET surface expression, intracellular signaling, ADC internalization and endolysosomal (EL) trafficking (using pHrodo Red-labeled Teliso-V), intracellular MMAE by LC/MS, and Teliso-V sensitivity were studied in 2D/3D cell cultures. Full-length c-MET protein structures were created with the ColabFold program and molecular dynamics simulation software (with/without artificial lipid bilayer) was used to predict the mechanistic basis for differential function.

Results: c-MET expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. Compared with WT-c-MET, various mutants showed oncogene-driven transformation evident by elevated levels of phosphorylated c-MET/AKT, cytokine-independent colony formation, oncogene-driven proliferation, and tyrosine kinase inhibitor (TKI) resistance. Interestingly, c-MET-TKD-expressing cells were the most sensitive to Teliso-V, which was associated with faster ADC internalization and EL trafficking. An AI-generated protein model and associated molecular dynamics simulation demonstrated that all oncogenic TKD variants tested stabilized the c-MET-plexin-semaphorin-integrin domain (R592-L614), known for its ability to position the extracellular ligand-binding (ELB) site for optimal activation.

Conclusions: c-MET-TKD mutations induced TKI resistance and enhanced MMAE delivery in association with robust ADC internalization and EL trafficking. Previous studies have shown that the HER2 ADC fam-trastuzumab deruxtecan-nxki was highly effective in cell lines and patients with ERBB2 kinase domain-mutant lung cancer via a similar mechanism, raising the possibility that this may be a general phenomenon across receptor tyrosine kinase-targeting ADCs. c-MET-TKD mutations are predicted to stabilize the ELB conformation to activate enhanced oncogenic signaling that may contribute to rapid endocytosis-mediated drug delivery.

Abstract 548: The microRNA-10b targeted therapeutic, TTX-MC138, is effective in preclinical pancreatic adenocarcinoma

Non-coding RNAs, such as miRNAs, represent promising targets for cancer therapy, given their role as critical players in oncogenic processes, including tumor initiation and progression, metastasis and therapeutic resistance. We and others demonstrated that miRNA-10b is a critical driver of tumor cell viability, especially in the metastatic setting. Based on this discovery, it became feasible to treat established metastatic cancer by inhibiting miR-10b. This was accomplished using a therapeutic, termed TTX-MC138, composed of antagomirs against miR-10b, conjugated to dextran-coated iron oxide nanoparticles. In our previous studies in murine models of metastatic breast cancer, we demonstrated the effectiveness of TTX-MC138 at preventing and eliminating existing metastases. Here, we describe the application of TTX-MC138 for the treatment of pancreatic adenocarcinoma. TTX-MC138 was administered weekly via intravenous injection into mice bearing orthotopic xenografts derived from human pancreatic adenocarcinoma cells. Tumor burden was measured by in vivo bioluminescence imaging. Animal survival, body weight, and tumor and metastatic burden at necropsy were also analyzed. Serum and tumor miR-10b expression, as well as global gene expression using RNA-seq were assessed to provide insight into target engagement and molecular function. Animals treated with vehicle or gemcitabine served as controls. Tumor growth rate was found to be significantly lower in animals treated with TTX-MC138 vs controls (p < 0.0001 for TTX-MC138 vs. PBS and vs. gemcitabine). Importantly, 40% of the animals treated with TTX-MC138 regressed their tumors completely as measured by bioluminescence imaging. Treatment continued for 10 weeks, after which the responding mice were monitored for disease recurrence without treatment for an additional 10 weeks. The animals showed evidence of continued weight gain and durable survival. They were sacrificed 10 weeks after treatment was discontinued, despite no evidence of morbidity, to perform necropsy. No tumors or metastases were found at the time of sacrifice. Gene expression analysis confirmed target engagement and molecular mechanism of action. Against the background of our earlier work, these studies provide further evidence of the potential of TTX-MC138 for cancer therapy and expand its relevance beyond metastatic breast cancer to also include pancreatic adenocarcinoma.

Abstract 556: Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression

An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC50 values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC50 values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p<0.0001) or 40H3-MMAE (p<0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p<0.0001) and 26% with 40H3-MMAE (p<0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs.

Abstract 558: EGFR inhibition in lung adenocarcinoma upregulates cell surface expression of the placental antigen ALPP and enhances efficacy of ALPP-ADC therapy

Expression levels of placental alkaline phosphatase (ALPP) and ALPP-like 2 (ALPPL2) are relatively low in most cancer types, limiting potential benefits from ALPP or ALPPL2 targeting therapies. Enhancing their expression would be an attractive approach for targeted therapy. We have undertaken analysis of ALPP and ALPPL2 protein expression in whole cell extracts (WCE) and the surfaceome of 158 cancer cell lines and found ALPP, and to a less extent ALPPL2, to be expressed on the surface at relatively low levels across multiple cancer types. We explored various means to enhance ALPP expression in lung adenocarcinoma (LUAD) and found that induction of cancer cell quiescence via nutrient deprivation, or treatment with EGFR inhibitors, greatly enhanced ALPP surface expression. Mechanistic studies revealed that enhancement of surface ALPP expression in LUAD cells following gefitinib treatment was mediated through repression of MEK/ERK signaling and activation of the transcription factor FoxO3a, which was identified as an upstream transcriptional regulator of ALPP. Using xenograft models of LUAD, we further demonstrated that gefitinib treatment upregulates surface expression of ALPP in LUAD cells but not in normal tissues. Combination therapy with gefitinib and an ALPP antibody conjugated with Monomethylauristatin F resulted in enhanced tumor suppression compared with gefitinib alone. Our findings support a novel combination treatment modality that boosts the efficacy of ALPP-ADC directed therapy.

Abstract 559: Expression of molecular targets for antibody-drug conjugates in patients with NSCLC and RET fusions

Background RET fusions are found in 1-2% of patients (pts) with advanced non-small cell lung cancer (NSCLC). RET inhibitors are highly effective, but resistance eventually occurs. Antibody-drug conjugates (ADC) are promising treatments in NSCLC, that could be employed at time of resistance. Data on the membranous expression of ADC targets, such as EGFR/HER2/HER3 and the possible impact of novel therapeutic agents in RET+ NSCLC are lacking.

Methods We conducted a single-center observational study, collecting tissue specimens from 19 patients (pts) affected by RET+ NSCLC. The expression of EGFR/HER2/HER3 proteins on the cellular membrane was evaluated by immunohistochemistry (IHC). EGFR expression was assessed by using the DAKO EGFR pharmDx kit (Dako, Glostrup, Denmark); HER2 staining (4B5 clone by Roche clone) was evaluated using ASCO/CAP guidelines in scale from 0 to 3+; HER3 staining (D22C5 clone by Cell Signaling) was categorized by intensity as 0, 1+, 2+, and 3+, according to the same criteria used for HER2 evaluation. All IHC analyses were performed at Gustave Roussy.

Results Out of 19 pts, 18 were evaluable for EGFR expression, 18 for HER2, 17 for HER3. All analyses were conducted on archival tissue samples from tumor biopsies performed predominantly in treatment-naïve pts (17/19). Pts had adenocarcinoma in 5/19 cases, KIF5B fusion partner in 13/19 cases. Median PD-L1 expression in evaluable pts was 15% (IQR 1- 60.00). EGFR expression was high, moderate, and low in 4 (22%), 2 (11%) and 5 (27%) cases, respectively. HER3 expression was 2+ in 4 (24%) cases, 1+ in 5 (29%) pts and 0 in the other 47%. None had HER2 membranous expression. EGFR expression was more frequently found in pts with uncommon fusion partners (6/11 vs 0/7, p=0.038) and non-adenocarcinoma histology (5/11 vs 0/7, p=0.10). HER3 expression evaluated at 2+ were identified in 3 cases with KIF5B-RET fusion and adenocarcinoma and 1 case with ANKRD26-RET fusion and undifferentiated histology. PD-L1 expression did not show significant difference between pts with positive vs negative EGFR or HER3 staining (p=1).

Conclusions EGFR and HER3 had variable expression in RET+ NSCLC, deserving further exploration for novel treatment strategies. HER2 expression was not found in our RET cohort.

Abstract 560: Disitamab vedotin, an investigational HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical cancer models

Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2, an oncogenic growth factor receptor that promotes cell proliferation and survival. DV consists of a novel anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable linker. DV has multimodal antitumor mechanisms of action that include direct cytotoxicity of HER2-expressing cancer cells and bystander effect based-cytotoxicity of neighboring cells, both of which are mediated by the intracellular release of MMAE within the targeted cell. Released MMAE can induce immunogenic cell death (ICD), which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity (ADCC), which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth and proliferation. In this preclinical study, we investigated the antitumor activity of DV in breast and gastric cancer models, both as a monotherapy and in combination with tucatinib, a HER2-selective oral tyrosine kinase inhibitor approved in combination with trastuzumab and capecitabine for patients with HER2+ metastatic breast cancer. In vitro, DV demonstrated cytotoxic activity against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range, and was more potent than the HER2-directed ADC trastuzumab emtansine (T-DM1). Internalization assays using time-lapse microscopy of breast cancer cells continuously exposed to labeled naked antibodies showed that disitamab internalized to a greater magnitude than trastuzumab. In a subset of those cell lines, DV showed enhanced internalization compared to other HER2-targeted ADCs, namely T-DM1 and trastuzumab deruxtecan (T-DXd). Similar internalization and cytotoxic activities of DV were observed in gastric cancer cell lines. We explored whether dual HER2 targeting with DV in combination with tucatinib improved the antitumor outcomes. In vitro results demonstrated evidence of enhanced cytotoxicity over single agents when tested in breast and gastric cancer cell lines with a wide range of HER2 expression levels. Investigation of the mechanism of the enhanced cytotoxicity revealed increased DV internalization in the presence of tucatinib, attributable to elevated HER2 levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast and gastric cancer settings as a monotherapy or in combination with tucatinib.

Abstract 562: Anti-tumor efficacy of GMF-1A3, an MMAE-based antibody drug conjugate targeting cell surface cleaved Amphiregulin in breast cancer

The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we describe the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. A pan-cancer tissue microarray analysis indicates that the target is commonly detected (> 50% of cases) in breast, prostate, liver and lung cancer.

Abstract 563: Preclinical development of YL202, a novel HER3-targeting antibody-drug conjugate (ADC) with novel DNA topoisomerase I inhibitor for treatment of solid tumors

HER3, a member of the HER family, is over-expressed in cancer cells and further elevated after HER2/EGFR/endocrine targeting treatment. YL202 is a novel HER3-targeting antibody-drug conjugate (ADC) structurally composed of a human anti-HER3 antibody, protease-cleavable linker, and a novel topoisomerase I inhibitor. This ADC was prepared using MediLink’s TMALIN platform, a proprietary tumor microenvironment activable linker-payload platform, which achieved a high drug-to-antibody ratio through homogeneously conjugated and favorable hydrophilic linker-payload.

In preclinical studies, YL202 exhibited strong reactivity, highly internalization and potent cytotoxicity toward tumor cells. YL202 also demonstrated significant dose-dependent antitumor activity in cell line- and patient-derived xenograft (CDX and PDX) mouse models representing several cancer types and could induce complete tumor regressions with no observable toxicity. YL202 showed stable PK profile with overlapping ADC and TAb curves in a 28-day cynomolgus monkey study. GLP toxicity studies using cynomolgus monkeys showed that YL202 is well tolerated with calculated therapeutic index (TI, HNSTD/MED) of ~100 for repeat dosing. No lung or platelet toxicity findings were observed at doses up to the maximum tolerated dose (MTD).

Based on these preclinical results, it demonstrates that YL202’s advanced ADC design results in an increased therapeutic margin, and YL201 could be further developed in HER3 positive cancer patients.

Abstract 583: AVA3996, a novel pre|CISION™ medicine, targeted to the tumor microenvironment via fibroblast activation protein (FAP) mediated cleavage

AVA3996 is a therapeutic product based on proprietary pre|CISION™ technology which incorporates a substrate that is sensitive to cleavage by FAP. The pre|CISION™ substrate can be utilized in a drug conjugate linker or to generate chemotherapeutics that are only activated in the tumor microenvironment. AVA3996 consists of a proteasome inhibitor molecule covalently bonded to a peptide containing a cleaving sequence (D-Ala-L-Pro), which is designed to be susceptible to hydrolysis by Fibroblast Activation Protein α (FAP) but is resistant to hydrolysis by both closely related and wider mammalian peptidases. FAP, a post-prolyl endopeptidase, is overexpressed on the surface of activated fibroblastic cells which are abundant in the supporting stroma of over 90% of malignant epithelial cancers.

Proteasome inhibitors are a first line of treatment for certain hematologic indications such as multiple myeloma. One consequence of proteasome inhibition is inhibition of the NFκB pathway due to reduced cleavage of the inhibitor IκBα; this pathway is activated in many tumors and represents an attractive target for a range of indications.

However, clinical utility of proteasome inhibitors is limited by severe dose-limiting toxicities, including peripheral neuropathy. AVA3996 has the potential to deliver elevated, effective levels of proteasome inhibitor directly to the solid tumor microenvironment while reducing systemic exposure and hence associated toxicities.

We have demonstrated that the pre|CISION™ substrate is exquisitely sensitive to FAP and is not cleaved by related proteases. The active AVA3996 warhead should therefore be released primarily following FAP cleavage in the tumor microenvironment.

Cancer cell lines were assessed for their sensitivity to AVA3996 in vitro: activity was typically 100-fold less than the active warhead or Bortezomib alone. Upon co-incubation with soluble FAP, potency of AVA3996 increased to similar levels as seen for the warhead alone demonstrating the masking effect of the pre|CISION™ substrate. A DRF/MTD study established the maximum tolerated dose in rats was around 6-fold higher for AVA3996 compared to warhead alone. This provides further evidence that AVA3996 can be dosed at higher levels than proteasome inhibitor alone, with the potential for greater tumor targeting and hence reduced systemic toxicity.

Ongoing in vivo and co-culture studies aim to further validate the efficacy and tolerability profile of AVA3996 to direct future development of this drug. In addition, the data supports wider utility of the pre|CISION™ platform to target therapeutics to the tumor while reducing systemic dose-limiting toxicities.

Abstract 596: Development and assessment of a novel tumor microenvironment activable linker (TMALIN) ADC platform for solid tumor treatments

Antibody-drug conjugates (ADC) are one of the fastest growing anticancer drugs, which bring cytotoxic drugs into tumors while avoiding high systemic toxicities. To date, all commercial ADCs use intracellular lysosomal cleavage mechanisms for payload release to kill tumor cells which is often limited by the low tumor antigen expression. To this end, a novel systemically stable and extracellular tumor microenvironment activable linker-payload technology platform has been developed.

Preclinical assessments demonstrated that the highly hydrophilic linker-payload, which showed limited impact on the hydrophilicity of antibody, prolonged the retention time in vivo and eventually enhanced the in vivo potency of ADC. The linker-payload is stable with less than 2% payload drop-off after 28-day incubation in plasma under physiological conditions in vitro. The high stability was also illustrated by GLP cynomolgus monkey studies and preliminary clinical trial data, with the overlap of ADC and TAb PK profile. Furthermore, a battery in vitro and in vivo cell line-derived xenograft (CDX) mouse model studies showed the linker-payload is stable in normal tissues but efficiently releases the payload in tumor tissues. The ADCs using TMALIN platform elucidated significant anti-tumor activity advantages when compared with those marketed ADCs targeting the same antigen, besides showing an acceptable safety profile in pivotal NHP studies without lung/liver/kidney toxicities.

In summary, the TMALIN platform addresses the unmet need of ADCs by releasing the payload extracellularly in tumors and tumor microenvironments on top of the known intracellular lysosomal cleavage mechanisms. Continued clinical validation work is underway for several pipeline candidates as well as investigation into other target areas.

Abstract 687: Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis

ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2, developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibits mammary carcinoma growth and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system: Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administered alone, thanks to the intrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16 (FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fat pad with 1 million QD cells; vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas 70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free one year after cell challenge. Mice challenged intravenously (i.v.) developed more than 300 lung metastases, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 and develop aggressive mammary carcinomas with a median latency of 5 months. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lung nodules, whereas 73% of mice therapeutically vaccinated without adjuvant, and all mice vaccinated with E2SB-C001+ISA 51, were free from metastases. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL in Delta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++ breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated by ELISPot (21±3 spots/2x105 cells). The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2.

Abstract 814: Novel strategy for aptamer-directed nanovesicle targeting in cholangiocarcinoma

Background: Cholangiocarcinoma (CCA) is a heterogeneous malignancy arising from the biliary epithelium. Its diverse molecular landscape and aggressive biology render many anti-cancer therapies ineffective. Nanovesicle technology provides an opportunity for therapeutic inhibition of oncogenic targets that have been previously classified as undruggable. EpCAM is an epithelial-specific, transmembrane glycoprotein with increased expression in human and murine CCA which can be used for nanovesicle targeting. As a proof of concept study, we designed and validated a novel strategy to direct therapeutic milk-derived nanovesicles (tMNVs) to CCA tumors.

Methods: tMNVs were decorated with RNA nanoparticles containing a validated aptamer (EpDT3) against EpCAM conjugated to a cholesterol-triethylene-glycol (TEG) scaffold containing an Alexa647 fluorophore. Human and murine CCA cell lines were treated with aptamer directed tMNVs and assessed for nanovesicle uptake by fluorescent microscopy. CCA tumor tissue, derived from orthotopic implantation of a syngeneic CCA cell line, SB1, into a C57BL/6 mouse, was collected and treated with either aptamer-directed or bare tMNVs ex vivo, and compared with adjacent normal liver tissue. Flow cytometry was utilized to characterize tMNVs absorption profile. C57BL/6 mice who had previously undergone SB1 orthotopic and flank implantation were treated with aptamer-directed tMNVs by tail-vein injection and subsequently euthanized. Tissue was collected for biodistribution analyses by fluorescent microscopy. The experiment was repeated in NOD-scid mice following orthotopic implantation of patient derived xenograft (PDX) CCA tumor.

Results: Both human and murine CCA cells treated with aptamer-directed tMNVs demonstrated high fluorescent signal consistent with tMNV absorption within 12 hours of application. Flow cytometry analysis showed aptamer-directed tMNVs were absorbed at a higher proportion by CCA tumors than bare tMNVs ex vivo. Aptamer-directed tMNVs also had better absorption by CCA tumors compared to adjacent normal liver tissue. Following treatment with aptamer-directed or bare tMNVs in vivo, fluorescent microscopy demonstrated that aptamer-directed tMNVs were significantly better absorbed in the orthotopic SB1 tumors, followed by the subcutaneous tumors. Minimal fluorescent signal was noted in the normal adjacent liver. Orthotopically implanted PDX tumors also demonstrated high fluorescent signals following intravenous treatment with aptamer-directed tMNVs.

Conclusions: Utilizing a novel targeting strategy, we were able to design tMNVs capable of reliably and specifically targeting CCA in preclinical models. This work is foundational to the future application of nanovesicle technology in the CCA treatment paradigm.

Abstract 816: Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models

Purpose: This study aimed to improve the systemic stability and enhance the anticancer activity of gemcitabine (Gem) by modifying Gem with stearic acid to form 4-N-stearoylGem (4NSG). The 4NSG compound was formulated into solid lipid nanoparticles and tested against patient-derived pancreatic cancer (PCa) cell lines and patient-derived xenograft (PDX) mice bearing subcutaneous tumors.

Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was characterized using high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and elemental analysis. 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using a particle size analyzer. Patient-derived primary PCa cells (PPCL-46 and PPCL-68) were treated with GemHCl and 4NSG-SLN. Cytotoxicity, cell migration, and cell-cycle studies were performed to determine the effectiveness of 4NSG-SLN against primary PCa cell lines. We performed antitumor efficacy testing using GemHCl and 4NSG-SLN in PDX mice bearing subcutaneous pancreatic tumors.

Results: Analysis of the H-NMR spectra displayed an amide bond at 10.88 ppm, confirming the conjugated bond between the 4-amino group of Gem and stearic acid. The purity of 4NSG was 99.8%. The hydrodynamic diameter (particle size) of 4NSG-SLN was 82±2.3nm, while the blank nanoparticle (SLN) was found to be 35 ± 4.3 nm. The half-maximal inhibitory concentration of 4NSG-SLN-treated PPCL-46 (IC50 =12 ± 2.1µM) and PPCL-68 cultures (IC50 =22 ± 2.6µM) showed higher cytotoxic activity compared with GemHCl-treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p<0.001). Our migration study showed 4NSG-SLN treated PPCL-46 cells at 5μΜ concentrations significantly reduced cell mobility towards the wound area with (28 ± 3.5) number of cells migrated compared to GemHCl treatment which showed (120 ± 3.8). The cell-cycle analysis showed that 4NSG-SLN treated PPCL-46 cells at 5μΜ concentration had a higher G1 population (78.25%) than GemHCl (72.75%). 4NSG-SLN may have triggered apoptosis in PPCL-46 cells (2.45%) at 20μΜ in the S and G2/M phases compared to GemHCl treatments which showed a higher cell population of (9.54%).4NSG-SLN treated PDX mice exhibited a two-fold decrease in tumor growth compared to GemHCl-treated PDX mice bearing tumors.

Conclusion: This study reveals that 4NSG-SLNs may potentially prolong Gem's systemic circulation, increase its bioavailability, and, most importantly, enhance the therapeutic efficacy of Gem in the treatment of PCa.

Abstract 817: Effective near infrared laser-induced photothermal therapy for cancer using indocyanine green/methyl-Β-cyclodextrin compound loaded liposomal nanoparticle

Background: Photothermal therapy (PTT) is a promising cancer therapeutic process to eliminate solid cancer through thermal ablation using electromagnetic radiation and photothermal agent (PTA). PTT is efficient in treating tumor because tumor has a lower heat resistance due to the shortage of blood supply. Also, it has less invasiveness, low toxicity to normal tissues, high spatial specificity, and minimal drug resistance. Indocyanine green (ICG), an FDA approved agent, is a widely used as a PTA and imaging probe in clinical application.

However, ICG has several major shortcomings that may hinder its practical use such as short half-life in circulatory system, unspecific binding to protein, and rapid decomposition by heat, light and aqueous solution, resulting in a decrease in PTT efficacy. To overcome these problems, we developed ICG/methyl-beta-cyclodextrin (MB-CD) compound-loaded liposomal nanoparticle by host-guest complexation between ICG and MB-CD and its liposome formation. It was possible to improve ICG stability and anti-tumor efficacy.

Method: Liposomes were synthesized using 1,2-Distearoyl-sn-glycero-3-phosphoethanolamone-N-(methoxy-(polyethylene glycol)-5000), 1,2-Distearoyl-sn-glycero-3-phosphocholine, cholesterol, methanol, and chloroform by film hydration method. To form ICG/MB-CD compound (IDC), ICG and MB-CD solution were mixed and purified. Then it was loaded into liposomal nanoparticles and filtered to prepare ICG/MB-CD compound-loaded liposome (IDL).

Results: We compared ICG, IDC, indocyanine green loaded liposome (ICL), and IDL. IDC and IDL were characterized by absorbance spectra, fluorescence spectrometer, dynamic light scattering, and transmission electron microscopy. IDL was less decomposed in various solvents than others, indicating of higher in vitro stability. Moreover, IDL had an enhanced fluorescence intensity among the four groups due to the protection of ICG conjugated structure by MB-CD. In addition, it was remarkable for IDL to be the least bleached by near infrared laser compared to others for demonstrating its better photothermal stability and retention ability. In confocal imaging and in vitro PTT experiment using 4T1 breast cancer cell line, IDL was well uptaken by the cells and showed the highest cancer therapeutic efficacy. Furthermore, in vivo and ex vivo fluorescence imaging were performed using 4T1 tumor bearing mice after intravenous injection of the particles. As a result, IDL injected group demonstrated the highest tumor uptake among the four groups.

Conclusions: IDL was successfully synthesized and exhibited excellent optical property, stability, and anti-tumor efficacy. Thus, it could be a promising PTA for treatment of solid cancer.

Abstract 821: Targeting metastatic prostate cancer using planetary ball-milled (PBM) nanoparticles

Men are most likely to develop prostate cancer (PCa), which has a high morbidity and fatality rate. Chemotherapy, the most recommended therapeutic strategy to combat PCa, has shown adverse effects on patients and develops drug-resistant PCa resulting in overall restricted survival of the patients. Many studies have demonstrated that natural compound-mediated therapy can be an effective option for treating PCa. Among the natural compounds, Thymoquinone (TQ), a derivative of Nigella sativa, has shown anti-proliferative, antioxidant, and anti-cancer effects. However, issues remain regarding its bioavailability, safety, and effective delivery system to cancer cells in vivo. In this study, we developed TQ nanoparticles (NPs) alone or in combination with DTX-encapsulated planetary ball-milled nanoparticles (PBM-NPs), formulated with starch (natural polysaccharides) inner core and biodegradable co-polymers poly (ε-caprolactone)/poly (ethylene glycol), functionalized with a prostate-specific membrane antigen (PSMA) aptamer (A10) to target the metastatic niche in PCa metastasis pre-clinical (patient-derived xenograft (PDX) and intra-tibial metastatic (LNCaP, PC-3 cells were injected directly into the tibia of SCID mice.) models. To determine the minimum effective dose (MED), efficacy, and safety of PBM-NPs, mice were treated intravenously with dose levels of vehicle control (empty-PBM-NPs), DTX alone (5 mg/kg, weekly), TQ alone (10 mg/kg, weekly), and a combination of TQ and DTX (25-fold less than maximum tolerance dose) using PBM-NPs, respectively for five weeks. Blood serum chemistry for toxicity and immunohistochemistry were performed to characterize these metastatic models. Our results showed TQ+DTX PBM NPs suppressed p-EGFR, ERK1/2, ABCB1, and Caspase3-expression, which are regulated by the EGFR-dependent pathway. We detected OPG, RANKL, PTHrP, and ET-1 immunoreactivity in all xenografts in the bone. In conclusion, sustained-release of combination PBM NPs demonstrated more significant anti-tumor activity following their conjugation to a prostate cancer cell-specific ligand, the toxicity of the drug can be reduced, and effective concentrations of the drug can be maintained locally for prolonged periods and prevention of PCa bone metastasis.

Abstract 825: Targeted nanospheres for the treatment of cancer

Conventional cancer therapy exploits sensitivity of dividing cells to DNA damage leading to apoptosis and tumor kill. Off-target normal tissue toxicity limits the maximum tolerated dose (MTD) and potential tumor ablation. We developed a targeted nanoparticle therapeutic platform that maximizes delivery of small molecules to tumor cells, including across the blood-brain barrier that normally prevents effective therapy with such agents. The core technology is a novel nanoparticle formulation that maximizes bioavailability and preferentially targets tumor cells while minimizing delivery to normal cells. Plasma irinotecan levels remain readily detectable for at least 48 hours, while free irinotecan is no longer detectable at 2 hours. Nanoparticles are readily imaged in tumor cells when virtually none are detectable in normal tissues. Targeting is achieved with antibodies that cause the nanoparticles to preferentially bind to tumor cells versus normal by a factor of at least 10. Levels of drug by quantitative mass spectroscopy are at least 15-fold greater in tumor vs normal tissue. As a result, the minimum effective dose (MED) is typically reduced ten-fold. Typical payloads include irinotecan, doxorubicin, and gemcitabine. Targeting antibodies include CD276 (B7H3) and CD99 (MIC2) for solid tumors. Compared to antibody-drug conjugates (ADCs), far greater drug doses per antibody molecule are delivered. In addition, the nanoparticles readily pass through the blood-brain barrier at least 15 times better than free drug alone. Consequently, the platform technology shows promise for treatment of both extra-cranial and intra-cranial tumors. In pre-clinical animal studies, survival of human PDX glioblastoma (GBM) bearing mice was prolonged by at least three-fold compared to free-drug treated animals when treated at a sub-optimal dose of 10mg/kg of irinotecan thrice weekly. Ewing sarcoma bearing mice showed indefinite survival at 2 mg/kg bi-weekly dosing, even for relapsed, treatment-resistant tumors. Treatment of hepatocellular carcinoma bearing mice biweekly at 5mg/kg resulted in no detectable tumor and 100% survival at 80 days, when all free irinotecan treated mice at the same dose were dead, with control animals dead at 50 days. Similarly, prostate cancer bearing mice treated at the same dose and regimen were all alive at 58 days when all untreated tumor bearing mice were dead. A similar result was obtained with ovarian cancer and the same treatment regimen, but tumor growth in treated animals was observed at 58 days when all untreated animals were dead. Perhaps most significantly, treated mice bearing pancreatic cancer showed 100 % survival at 80 days when all untreated animals were dead at day 59. At no time did any animal show evidence of treatment-related toxicity. Taken together, these results support a non-toxic cancer treatment approach with existing small molecule therapy that preferentially delivers drug to tumor with markedly improved efficacy.

Abstract 828: DNA origami nanostructures as a targeted payload delivery system

DNA origami is a platform nanotechnology allows for the generation of nanostructures with precisely defined geometric shape that may be easily functionalized with a variety of payloads for therapeutic delivery. These payloads include nucleic acids (miRNA, siRNA, gene sequences), associated Cas protein molecules, therapeutic small molecules drugs, functional peptides (adjuvants or cell penetrating peptides), and incorporated targeting and/or therapeutic antibodies. Hence, DNA origami is a highly promising vehicle to deliver targeted therapeutic payloads to cancer cells or other diseases. Previous studies by our laboratory and others revealed DNA origami mediated drug delivery efficiently delivered daunorubicin or doxorubicin to cancer cells while outperforming free drug in both solid and liquid tumor model systems in vitro and in vivo. Our recent findings showed significant survival advantage by doxorubicin-loaded DNA origami over delivery of free doxorubicin in an aggressive, acute monocytic leukemia model. We also showed antibody-directed drug delivery by DNA origami may be directly targeted to monocytic leukemia cells expressing the target surface antigen, and superior efficacy in vitro against acute myeloid leukemia cells when loaded with daunorubicin relative to free drug at low concentrations. Thus, antibody targeted drug loaded DNA origami represent a promising novel precision medicine approach. Additionally, recent findings by our laboratory in vivo determined that DNA origami nanostructures alone administered at high dose (12.0mg/kg) distribute well, are non-toxic, and illicit mild immunogenicity, making them an attractive candidate for further development towards therapeutic applications. We recently developed a DNA origami structure functionalized at a high density with adjuvant (CpG) and antigenic peptides (OVA) to stimulate a directed antigen-specific immune response. We have shown this DNA origami vaccine platform displays significant improvement in efficacy by an OVA-pulsed antigen-killing assay in vivo relative to alum and CFA standard adjuvant formulations. Thus, our DNA origami vaccine platform represents a promising novel approach to optimize an anti-cancer immune response, a finding also shown by other laboratories.Collectively, DNA Nanobot technology offers 1) a promising a cancer drug delivery system that can deliver a high number of therapeutic drugs (100s of drug molecules per targeted delivery system) utilizing existing ADC conjugation methods; 2) An optimal vaccine delivery method to enhance efficacy; and 3) cell specific targeted delivery of therapeutic nucleic acids and genes to modify oncogene and/or tumor suppressor gene expression.

Abstract 837: Preclinical development of ELU001: A folate receptor alpha (FRα)-targeted C’Dot drug conjugate (CDC) for the treatment of brain metastases

Antibody-drug conjugates (ADCs) exhibit limited efficacy against brain malignancies due to their inability to cross the blood-brain barrier (BBB) and penetrate into solid tumors. In clinical imaging studies C’Dots readily crossed tumor-disrupted BBB and penetrated across/localized in tumors in the brain avoiding healthy brain. CDCs are ultra-small (6-7 nm) nanoparticles with a silica core coated by short polyethylene glycol chains conjugated to up to 80 small payload and targeting moieties, creating highly potent and avid agents. CDCs’ small size mediates rapid renal clearance leading to limited exposure to healthy tissues.

ELU001 is a potent anti-FRα CDC with an average of 21 exatecan topoisomerase-1 inhibitor payload molecules and 13 folic acid targeting molecules on its surface. ELU001 is currently in a dose escalation trial in patients with systemic solid tumor indications that have the potential to overexpress FRα. Early signs of activity with a manageable safety profile have been seen across antigen expression levels.

ELU001 exhibits in vitro potency in the sub/low nanomolar range against cancer cell lines that express 3+, 2+ or 1+ levels of FRα. In studies performed in immunodeficient mice bearing intracranial NCI-H2228-luc bioluminescent non-small cell lung cancer tumors, ELU001 was observed to penetrate across disrupted BBB and localize in tumors. Microscopic analysis revealed that ELU001 distributed throughout the tumors but not into the healthy brain. Treatment with a single cycle of ELU001 Q3Dx3 in mice bearing either early (Day 7) or late (Day 14) brain tumors was well tolerated and resulted in significant reduction in tumor burden measured by bioluminescence as well as a prolonged survival benefit. Retreatment of these animals resulted in an additional decrease in tumor burden. These results suggest that ELU001 may have promise for the treatment of metastatic brain tumors that are difficult to treat with currently available agents.

Abstract 960: Overlapping expression landscape of antibody drug conjugate targets, trophoblast cell surface antigen 2 (Trop-2) & human epidermal growth factor receptor 2 (HER2), in breast cancer

Background Antibody drug conjugates (ADC) are novel drugs linking potent payloads to antibodies targeting antigen-expressing tumors. Sacituzumab govitecan (SG), targeting Trop-2, is approved for metastatic triple negative breast cancer (TNBC); and trastuzumab deruxtecan, targeting HER2, is approved for HER2-positive and HER2-low metastatic breast cancer. To understand the potentially overlapping clinical landscape of Trop-2 and HER2 antigens, we evaluated RNA expression data in breast cancer from The Cancer Genome Atlas (TCGA) project.

Methods TCGA dataset was assessed for Trop-2 and HER2 expression via processed RNA sequencing (RNA-seq) data of the corresponding genes TACSTD2 and ERBB2. Medium/high gene expression was assessed as >100 transcripts per million (TPM). Samples were classified HER2-low per ASCO/CAP guidelines. Gene expression across clinical parameters was assessed via one-way ANOVA.

Table

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Results 1076 patients with primary breast cancer were included. The majority (59%) had both high TACSTD2 expression (TACSTD2hi) and high ERBB2 expression (ERBB2hi) (see Table). Median TACSTD2 expression was 572 TPM (IQR 349-666 TPM); median ERBB2 expression was 122 TPM (IQR 73-192 TPM). No significant difference was observed in TACSTD2 or ERBB2 expression among invasive ductal carcinoma, invasive lobular carcinoma, mixed histology, or other (p = 0.07, 0.23). No significant difference in TACSTD2 expression was noted between HER2-low and HER2-negative subtypes (p=0.34).

Conclusions While SG is approved in TNBC, TACSTD2 is expressed across all breast cancer subtypes, including HER2-low, suggesting a broader population may benefit from Trop-2-targeted ADCs. Furthermore, given that over half of breast cancers have high expression of both TACSTD2 and ERBB2, additional studies are needed to understand the optimal sequencing of ADC-based therapies for patients with breast cancer.Abstract 1133: A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression

Purpose: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. B7-H4 is over expressed in breast and ovarian cancer compared to normal tissues making it a promising target for therapies, however, to date it has not been evaluated if the expression is maintained in drug resistant cancer. Here we examine B7-H4 expression in high grade serous ovarian carcinoma (HGSOC) tumors at diagnosis and post-platinum or PARPi-resistance. We also evaluate the activity of a novel B7-H4-directed antibody-drug conjugate (ADC) bearing the pyrrolobenzodiazepine-dimer payload tesirine, in preclinical models of breast and ovarian cancer, including those resistant to standard of care therapies.

Experimental Design: B7-H4 expression was measured by quantitative flow cytometry and immunohistochemistry. ADC efficacy was tested against multiple cell lines in vitro and patient-derived xenografts (PDX) in vivo. The effect of ADC treatment on cell cycle, DNA damage, and apoptosis was measured using flow cytometry.

Results: B7-H4 was over-expressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels at multiple metastatic sites after acquired multi-drug resistance (n = 4 donors). Treatment with the ADC resulted in target-specific growth inhibition in a panel of ten B7-H4 expressing ovarian and breast cancer cell lines (IC50 6.18-273.9 pM, median 48.3 pM). Co-cultures of B7-H4 +/- lines support bystander killing effects leading to further tumor cell death in B7-H4- clones. In platinum- or PARPi-resistant ovarian cancer cells, ADC treatment significantly decreased viability and colony formation (P < 0.001) while increasing S or G2/M phase cell cycle arrest (P < 0.001) and DNA damage (P < 0.0001), ultimately leading to apoptosis (P < 0.01). A single 0.3 mg/kg dose of the ADC resulted in tumor regression in 61% of breast and ovarian PDX models tested (n = 23), where lower activity was identified in B7-H4 low or negative expressing models (P = 0.048). In PARPi- and platinum-resistant HGSOC models (n = 3), continuous B7-H4 ADC treatment (dosed once every 28 days) resulted in sustained anti-tumor activity, leading to complete (7 of 16) or partial responses (3 of 16) and increased survival (P < 0.004).

Conclusions: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC.

Abstract 1134: Therapeutic potential of an antibody-drug conjugate directed against a tumor-specific epitope on podocalyxin

Podocalyxin (Podxl) is a cell surface sialomucin that is frequently upregulated in tumors with high metastatic potential and its expression is associated with poor outcome in several human cancers. As such, Podxl is emerging as an important prognostic and theragnostic marker. While Podxl is expressed on normal vascular endothelia and kidney podocytes, we sought to produce a novel anti-Podxl antibody that selectively recognizes a tumor-restricted glycoepitope on the extracellular mucin domain of Podxl. The antibody we have produced, PODO447, is specific to the tumor glycoform of Podxl, demonstrated by a lack of binding to normal tissues that are known to express Podxl. In contrast, we show binding of the antibody to tumor cell lines, patient-derived cell lines, and primary tumor tissues. We have previously shown that the majority of tumors in an ovarian carcinoma array (219 cases), including 65% of the high-grade serous histotype, are positive for PODO447. Here, we further show the presence of PODO447 in tumors of the breast, urothelium, pancreas, endometrium, colon, prostate, lung (both small cell and non-small cell), and glioblastoma. To assess the therapeutic potential of our antibody as an antibody drug conjugate (ADC), we coupled PODO447 to the microtubule disruptor monomethyl auristatin E (MMAE) with an enzyme cleavable linker carbamoyl p-aminobenzyl carbamate (PABC), resulting in the ADC PODO447-Vedotin. We demonstrate promising in vitro activity of the ADC to various human tumor cell lines as well as in vivo efficacy to xenografted ovarian and pancreatic tumor lines. Our data reveals PODO447-Vedotin as a tumor-specific and highly efficacious therapeutic agent for the targeting of human tumors and as such, PODO447 exhibits potential for further development as a targeted clinical immunotherapy.

Abstract 1202: Targeting cell adhesion molecule 1 (CADM1) with an antibody drug conjugate for the treatment of osteosarcoma

Introduction: Survival outcomes for patients with osteosarcoma have been stagnant for decades. There is an urgent need to identify new surface targets in osteosarcoma and develop novel therapies to increase cure rates. Antibody-drug conjugates, due to their targeted delivery of cytotoxic payloads to specific targets, are an attractive class of agents for potential use in osteosarcoma.

Methods: We used an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Cell-surface expression of the identified target antigen was validated by IHC and flow cytometry in osteosarcoma cell lines, patient-derived xenograft (PDX) models, and a patient tumor tissue microarray. As a proof of concept, humanized CADM1 antibody (clone PTA021-A1) was conjugated with tesirine (SG3249), a pyrrolobenzodiazepine (PBD) dimer payload. The antitumor activity of this antibody-drug conjugate was tested in vitro and in vivo in osteosarcoma cell lines and PDX models.

Results: Cell Adhesion Molecule 1 (CADM1) was found to be enriched in osteosarcoma at both protein and mRNA levels, while minimally expressed in normal tissues. Flow cytometry validated the cell-surface localization and expression levels of CADM1 in 7 osteosarcoma cell lines. We performed immunohistochemistry (IHC) staining using an osteosarcoma tissue microarray from 37 patients and 19 PDX models. CADM1 was expressed in 100% of the patient samples and PDXs. The newly developed ADC induced cytotoxic effects in 100% (5/5) of osteosarcoma cell lines, with IC 50 at 120h ranging from 0.009 to 0.1 ug/ml. In vivo testing is ongoing. Preliminary data shows objective response and prolonged survival in selected osteosarcoma PDX models. The mice tolerated the ADC well with minimal toxicity.

Conclusions: CADM1 is highly expressed in most osteosarcoma samples and minimally expressed in normal human tissue, which makes it an attractive target for respective antigen-targeting ADC therapies. The CADM1 targeting ADC showed antitumor activities in osteosarcoma preclinical models which may warrant further investigation of CADM1 targeted therapies for osteosarcoma.

Abstract 1521: Unlocking the therapeutic index of highly potent site-specific ADCs in solid tumors

CD276 (B7-H3) is a transmembrane protein of the B7 family, which includes many prominent immune checkpoint targets, and is expected to play a role in T-cell stimulation, tumor-infiltration, as well as other tumorigenic functions with poor prognosis when upregulated. CD276 is highly expressed in solid tumors, cancer initiating stem cells, and tumor associated vasculature with minimal to no expression in healthy tissue. This promising expression profile has piqued interest for various targeted therapeutic modalities, such as antibody-drug conjugates (ADCs). Developing effective ADCs for solid tumors remains a challenge due to inefficient conjugation technologies which are either non-homogeneous, unstable, or do not position payloads at optimal sites. BrickADCs minimize hydrophobicity, optimize payload release kinetics, and tune biophysical properties to improve efficacy, safety, and bioavailability, resulting in improved therapeutic indexes. Potent payloads such as pyrrolobenzodiazepines have been underutilized due to toxicity, preventing their widespread use. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (unfettered site accessibility and chemistry), efficient, and scalable generation of ADCs which overcome these limitations. In this work, UAAs (unnatural amino acids) were incorporated into full-length anti-CD276 antibodies and conjugated with optimized pyrrolobenzodiazepine (PBD) payloads. BrickADCs leverage a proprietary Site-Select Panel to find optimal sites that shield the toxicity of highly potent, hydrophobic PBD payloads while maintaining their high activity to generate anti-CD276-UAA-PBD ADCs. The highest performing BrickADCs, developed by a rapid screen of PBD payloads conjugated to different sites in the antibody backbone, exhibit high therapeutic efficacy in a variety of CD276 positive solid tumor cell lines (eg. colon, ovarian, lung, sarcoma, etc) and in mouse xenograft models with a 3x differential in complete response. The tuned hydrophobicity and release kinetics have resulted in ADCs that have an increased therapeutic index, particularly due to their improved safety profile compared to cysteine conjugation.The BrickADC platform enables an increased therapeutic window for potent payloads across solid tumors, opening the floodgates for further development of ADCs which leverage payloads that have been plagued by poor therapeutic index. Continued work is underway for the CD276 pipeline candidates as they progress towards IND, as well as investigation into other targets and conjugate modalities.

Abstract 1522: SGN-B6A induces immunogenic cell death as an additional mechanism of action

SGN-B6A is a novel investigational antibody-drug conjugate (ADC) directed to integrin beta-6 and uses the clinically validated vedotin drug-linker platform that delivers the microtubule disrupting agent, monomethyl auristatin E (MMAE). SGN-B6A is designed to bind and internalize the integrin beta-6/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. We have previously demonstrated the antitumor activity of SGN-B6A in cell line-derived xenograft models originating from multiple carcinomas as well as patient-derived xenograft models of non-small cell lung cancer (NSCLC). Other clinically validated vedotin ADCs that deliver MMAE have been shown to induce immunogenic cell death (ICD) in preclinical models and have demonstrated promising clinical activity in combination with immunotherapy. Since the induction of ICD appeared to be a consequence of the activity of MMAE, and is independent of the antibody that delivers it, we hypothesized that this mechanism of action may also apply to SGN-B6A.

Consistent with this hypothesis, we observed that tumor cells treated with SGN-B6A in vitro showed key hallmarks of immunogenic cell death, including markers of endoplasmic reticulum (ER) stress, exposure of calreticulin, and release of ATP and high mobility group protein B1 (HMGB1). Further, in vivo studies demonstrated that treatment with SGN-B6A led to immune activation and recruitment of immune cells to the tumor environment. In an integrin beta-6-expressing syngeneic model, a vedotin ADC directed to integrin beta-6 has shown combinatorial activity with immunotherapy.

Preclinical models suggest that, like other vedotin ADCs, SGN-B6A induces immunogenic cell death which then promotes activation and recruitment of immune cells to the tumor. We have recently reported promising single-agent activity of SGN-B6A in non-small cell lung, head and neck squamous cell, and esophageal cancer observed in interim results of a phase I study (NCT04389632). The combination of SGN-B6A with immunotherapy may be utilized as a potential treatment for integrin-beta-6-expressing tumors including NSCLC, head and neck squamous cell carcinoma, and esophageal carcinoma. Altogether, our preclinical and initial clinical results support the ongoing evaluation of SGN-B6A as a single agent and in combination with immune checkpoint inhibitors.

Abstract 1523: Subcutaneous dosing of amatoxin-based ADCs increases the therapeutic index

Background: Amatoxin-based antibody-drug conjugates (so called ATACs) are a new class of ADCs that use the RNA polymerase II inhibitor amanitin as toxic payload. HDP-101 is the first ATAC in the clinic, currently being tested in a phase I/II clinical trial in multiple myeloma patients. HDP-101 is given to patients intravenously (i.v.) as this is the most common administration route for ADCs. I.v. is often selected as route of administration in a clinical setting since it results in a bioavailability of 100%. However, i.v. dosing has disadvantages such as very high Cmax levels which can trigger toxicity, requires hospitalization of the patient for administration, and bears the risk of vessel damage. In this study, we demonstrate that subcutaneous (s.c.) dosing is a promising alternative route of administration for ATACs which is well tolerated and able to improve the therapeutic index (TI) of ATACs.

Material and Methods: ATACs: ADCs based on cysteine-reactive and site-specific amatoxin-linker constructs synthesized at Heidelberg Pharma. Monkey Studies: Cynomolgus monkeys, escalating doses of ATACs i.v or s.c. q21d Mouse Studies: CB17 Scid mice, single dose of ATACs i.v. or s.c.; if applicable bleeding at 8 - 12 time points between 5 min and 14 days after dosing; tumor studies in s.c. and i.v. tumor models Results: The impact of the dosing route on organ distribution of ATACs was tested in a PK study after a single i.v. or s.c. ATAC dose. S.c. dosing resulted in increased serum half-life of ATACs and reduced Cmax values as compared to i.v. dosing, while the AUC was comparable. Toxicity of ATACs is most probably driven by its Cmax level in serum, whereas the efficacy is mainly driven by AUC. Thus, we hypothesized that s.c. dosing might improve the TI of ATACs. The maximal tolerated dose (MTD) of ATACs in mice was compared between s.c. and i.v. dosing. S.c. dosing resulted in higher MTDs as compared to i.v. dosing independent of the antibody and amatoxin payload variant used. These findings were also confirmed in cynomolgus monkeys, where s.c. dosing of the ATAC HDP-103 resulted in a reduced Cmax, a comparable AUC, and an improved HNSTD of HDP-103. The impact of s.c. dosing on anti-tumor efficacy of ATACs was investigated in several tumor models in mice. ATACs with different antibodies and amatoxin payloads were applied as single i.v. or s.c. dose. In all models tested, the anti-tumor efficacy of ATACs was similar between groups receiving s.c. or i.v. dosing.

Conclusions: The present study demonstrates that s.c. dosing is a promising route of administration for ATACs as it not only refines the pharmacokinetic distribution of ATACs but may improve the TI in patients. By reducing Cmax, s.c. dosing improves the tolerability of ATACs in mice and cynomolgus monkeys. At the same time the anti-tumor efficacy of ATACs is maintained as the AUC is not negatively impacted. Thus, s.c. dosing improves the TI of ATACs and may represent a promising route of administration also in humans.

Abstract 1529: Characterizing antibody internalization for rational selection of ADC linker design

Antibody-drug conjugates (ADC), which target a highly potent cytotoxin to a specific protein overexpressed on tumor cells, are a promising class of anticancer therapeutics. In addition to the antibody characteristics, intracellular reactivity of the antibody-cytotoxin linker (which affects drug release) is a key determinant of ADC efficacy. While many different linkers have been used in approved ADCs, the intracellular trafficking profile of an antibody will likely impact the effectiveness of different linkers differently. Here, we determined the cellular internalization of two antibodies, anti-perlecan antibody (‘AM6’), and anti-EGFR antibody (cetuximab; ‘Ctx’), in MDA-MB-231-LM2 cells (a lung-metastatic TNBC cell line). Using live-cell confocal microscopy, we investigated the effect of endocytosis inhibitors on intracellular trafficking of AM6 and Ctx. While AM6 was internalized quicker compared to Ctx, the internalization of both antibodies was reduced significantly by 5-(N-Ethyl-N-isopropyl)-amiloride, a macropinocytosis inhibitor and, to a lesser extent, by chlorpromazine, an inhibitor of clathrin-mediated-endocytosis. The results suggest both AM6 and Ctx were internalized by both macropinocytosis and clathrin-mediated endocytosis. We then investigated whether acidic pH in the lysosomes and the reductive environment in the endosomal vesicles would facilitate drug release from ADC. We conjugated thiol-reactive doxorubicin to reduced interchain disulfides of Ctx via either the acid and glutathione (GSH)-labile pyridinyldithio-hydrazide linker or the non-cleavable C6-maleimide linker. Intracellular doxorubicin release for the two ADCs was evaluated by live-cell confocal microscopy. Doxorubicin was successfully conjugated to Ctx, with a DAR of 4.8 and 8.6 for the cleavable and non-cleavable linkers, respectively. Conjugation of doxorubicin to the antibody was confirmed by fluorescent gel imaging after SDS-PAGE and by using hydrophobic interaction chromatography. Confocal microscopic studies showed that the ADC with cleavable linker resulted in greater doxorubicin accumulation in the nucleus compared to the ADC with non-cleavable linker, which was predominantly sequestered in the lysosomes. These results suggest that the cleavable linker results in better drug release compared to the non-cleavable linker for the Ctx-doxorubicin ADC. Studies examining the effect of linker chemistry on drug release from the anti-perlecan ADC and on the anticancer efficacy of the two ADCs are ongoing.

Abstract 1530: Gastric cancerantibody fragment drug-conjugates (FDCs): Applying a successful concept to solid tumours

Antibody Drug Conjugates (ADCs) have failed to deliver the successes seen in breast and urological tumours in Gastro-intestinal tumours such as stomach, colorectal and pancreatic due to 3 critical limitations: Low potency, ineffective solid-tumour penetration and poor tolerability. Approaches utilizing full-length Immunoglobulins dominate the industry, however, antibody fragments (e.g. single-chain Fvs-scFvs), which have many advantages including rapid tumour penetration, faster clearance, inexpensive manufacture, have been technologically challenging to apply in oncology. Our novel approach enables scFvs to have a high Drug:Antibody loading ratio (DAR) whilst retaining effective binding and other favourable biophysical properties, leading to a new product class tailored for solid tumours.

Antikor has two FDC products in development for solid tumours, notably gastric: anti-HER2 FDC (ANT-043) and a second target (ANT-045, to be disclosed). ANT-043 has pM potencies in a range of HER2-expressing cell-lines, including trastuzumab-resistant models, excellent tumour ablation effects in breast, ovarian and gastric cancer xenograft models and superior tolerability compared to an ADC in rat toxicology studies at a dose of 1mg/kg/weekly. Quantitative payload tumour uptake and fluorescent immuno-histological studies demonstrate superior solid tumour penetration across the entire tumour and diffusion from blood vessels. In collaboration with our partners, Essex Biotechnology PLC, Antikor is taking ANT-043 into development. ANT-045, which emerged from Antikor’s proprietary FDC ‘discovery engine’, is progressing towards IND-filing and updated data will illustrate how ANT-045 could have a broader patient benefit in gastro-intestinal cancers. ANT-045 has excellent in vitro cell-kill potency (pM IC50s) and excellent stability and superior in vivo tumour cure efficacy, compared to a leading clinical stage benchmark ADC. This presentation will focus on Antikor’s FDC discovery platform (stable high-DAR scFv-display libraries, tailored linker-payloads and design features) that has the potential to generate first-in-class products for difficult to treat solid tumours for patient benefit and promising to succeed where ADCs have failed to make an impact.

Abstract 1533: ZW220, a novel NaPi2b-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload

Background: NaPi2b is a multi-pass transmembrane sodium-dependent phosphate transport protein encoded by the gene SLC34A2. NaPi2b is involved in normal phosphate homeostasis and its expression is found in the lung, liver, and small intestine. NaPi2b is highly expressed in ovarian carcinomas, lung adenocarcinomas, and colorectal carcinomas. ZW220 is an antibody-drug conjugate (ADC) targeting human NaPi2b, wherein a humanized IgG1 antibody is conjugated to novel camptothecin-based topoisomerase I inhibitor, ZD06519. The drug linker in ZW220 is comprised of a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable sequence.

Materials and Methods: A series of in vitro and in vivo studies were conducted to interrogate the mechanism of action and the therapeutic potential of ZW220. The binding, internalization, potency, and bystander effect of ZW220 were evaluated in vitro using endogenous NaPi2b-expressing ovarian and lung cancer cell lines. In vivo, the anti-tumor activity of ZW220 was evaluated in a panel of cell line derived xenograft (CDX) models and ovarian patient derived xenograft (PDX) models featuring a range of NaPi2b expression. The pharmacokinetic (PK) profile of ZW220 was determined in Tg32 mice, a transgenic mouse expressing human neonatal Fc receptor (hFcRn).

Results: ZW220 antibody exhibited cross-reactivity to human and cynomolgus monkey NaPi2b, nanomolar binding affinity and rapid internalization in NaPi2b-expressing cell lines in vitro. ZW220 elicited target-specific, sub-nanomolar cytotoxicity in two-dimensional monolayer and three-dimensional tumor spheroid models and demonstrated bystander-mediated cell killing in cancer cell co-culture assays. The treatment of a panel of ovarian PDXs with a single dose of 6 mg/kg of ZW220 resulted in robust tumor growth inhibition. ZW220 demonstrated a favourable PK profile in Tg32 mice, with comparable half-life to its parental unconjugated antibody. These results support the potential of ZW220 as a novel therapeutic agent which may help address unmet medical need in patients with NaPi2b-expressing tumors.

Abstract 1538: Revisiting the dogma of antibody drug conjugates (ADCs): Emerging data challenge the benefit of linker stability and the primacy of payload delivery

Antibody drug conjugates (ADCs) are an effective class of cancer therapeutics which have become more prominent after eight FDA approvals in the past three years. ADCs are envisioned to enable the selective delivery of a potent drug to cells that express the antibody target while sparing normal tissues, and thus to improve the therapeutic window by both increasing the maximum tolerated dose (MTD) and decreasing the minimum efficacious dose (MED) of the conjugated drug. Mounting clinical evidence does not support this simplified paradigm [1]. Herein we focus on three prominent issues in ADC development:

1. Alternative mechanisms over and above direct tumor targeting are likely to contribute to ADC therapeutic benefits in the clinic [2]. The responses to certain ADCs in patients with different levels of target antigen expression reveal some target-independent antitumor effects, suggesting the possibility that circulating payload may contribute to efficacy.

2. The preclinical optimization of ADCs is based on efficacy and tolerability in non-human animal studies. Fundamental differences in biology and translatability between species potentially overemphasize the importance of linker stability.

3. Clinical data, although limited, does not support the idea that increased ADC stability drives substantial therapeutic benefit. None of the 13 approved ADCs are completely ‘stable’ in circulation, and more stable drug-linkers have been associated with alternative toxicity profiles and no significant improvement in MTD.

Novel meta-analyses and case studies assembled and integrated from >40 approved or clinically active ADCs and >70 discontinued ADCs will be presented in support of these three perspectives. The ADCs included in the analysis differ in terms of payload class, linker type, conjugation chemistry, drug-to-antibody ratio, and antibody target. Gaining a better understanding of the mechanism(s) of action of effective ADCs and the interrelationship between ADC structural components and their pharmacokinetics and pharmacodynamics is critical to inform the next generation of ADCs.

Abstract 1540: A click chemistry-activated auristatin protodrug (TCO-MMAE) demonstrates potency and safety with antibody and intratumoral tumor-targeting agents

Click chemistry encompasses chemical reactions that are highly efficient, rapid and proceed in complex environments. The 2022 Nobel prize in Chemistry was awarded to Sharpless, Meldal and Bertozzi in recognition of the transformative effect click chemistry has had in research and drug development. Using click chemistry to precisely control the activation of drugs at tumors, Shasqi has developed the Click Activated Protodrugs Against Cancer (CAPAC™) platform. In essence, a click chemistry activator is localized to tumors, which subsequently activates protodrug(s) bearing the companion click chemistry reaction partner. Shasqi is pioneering the use of click chemistry in the clinic. The lead candidate SQ3370 consists of an intratumorally (IT) injected tetrazine (Tz)-modified biopolymer and an intravenously (IV) administered trans-cyclooctene (TCO)-modified doxorubicin (Dox) protodrug. SQ3370 provides enhanced safety and efficacy in preclinical models compared to conventional Dox and is being evaluated in a Phase 2a study in soft tissue sarcomas (NCT04106492). We will present our research on an MMAE-based protodrug (TCO-MMAE), which is stable in plasma with dramatically attenuated activity in vitro and in vivo without the Tz activator. When treated with an activator in vitro, the potent cytotoxic activity of MMAE is restored. When paired with the Tz-biopolymer (IT), testing in the Karpas299 model of human non-Hodgkin’s lymphoma led to 6 of 8 (75%) complete responses in the test group. Treatment with TCO-MMAE (IV) + Tz-biopolymer (IT) also elicited significant tumor growth inhibition (p-value <0.01) in the RENCA model of murine renal adenocarcinoma. Modest body weight loss and transient cytopenia support the tolerability of the treatment. To enable systemic delivery of the click chemistry activator, we are developing a series of antigen-targeted conjugates to pair with the TCO-MMAE protodrug. We designed and generated a Fab-Tz conjugate that binds HER2. The conjugate had an average of 2.2 Tz per Fab and efficiently bound HER2-positive NCI-N87 cells. In a xenograft model of gastric cancer (NCI-N87), the Fab-Tz was dosed IV, followed by the TCO-MMAE protodrug (IV). Treatment with HER2 Fab-Tz + TCO-MMAE induced significant tumor regression compared to vehicle while a negative control Fab-Tz + TCO-MMAE treatment did not (p-value <0.05). To date, we have demonstrated the versatility and modularity of the CAPAC platform. Anti-tumor activity was observed in multiple preclinical models for the TCO-MMAE protodrug in combination with both biopolymer and Fab targeting agents. These results highlight the power of click chemistry to activate a protodrug at the tumor site, independent of tumor biology, the molecular format or delivery method of the targeting agent.

Abstract 1541: NextGen conditionally active biologic (CAB) anti-Nectin-4-ADC with improved stability and safety

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents. Today, development of a new ADC faces two major challenges: on-target, off-tumor toxicity based on target expression on healthy tissue and off-target, off-tumor toxicity caused by extracellular linker vulnerability and/or premature payload release. On-target, off-tumor toxicity can be effectively eliminated by using a Conditionally Active Biologics (CAB) antibody to specifically target tumor cells. CAB technology is a proprietary platform used to engineer antibodies that target antigens in the context of diseased tissues, but not normal tissues by exploiting the unique extracellular acidic conditions found on the surface of cancer cells (Chang et al. 2021). This platform has allowed us to generate CAB antibody drug conjugates (ADCs) specific to tumor associated antigens that are often expressed on healthy tissues. CAB selectively reduces on-target toxicity, as well as off-target toxicity by eliminating target mediated drug disposition. Conventional ADCs can induce unacceptable toxicities caused by binding to antigen expressed on normal cells, resulting in the undesired release of the payload. This can severely limit the therapeutic window of the drug candidate. Two CAB-ADCs (Mecbotamab Vedotin and Ozuriftamab Vedotin) using the well-established vedotin linker with MMAE as payload are currently in phase 2 clinical trials and show promising results with increased safety profiles, with currently no signs of on-target, off-tumor toxicity. BA3361, a CAB-ADC targeting Nectin-4, showed good in vitro and in vivo efficacy data demonstrating cytotoxicity against human Nectin-4-expressing tumor cell lines and tumor regression in xenograft models using the vedotin linker (AACR 2020, Poster #4560). To eliminate vedotin linker-related off-target toxicities, we developed a NextGen Nectin-4 CAB ADC with a novel cleavable linker that is highly stable in human serum and has increased solubility. In vivo efficacy data from CDX models as well as data from non-GLP tox studies in rats and cynomolgus monkeys will be presented. In conclusion, our data indicates that the CAB-Nectin-4-ADC with its optimized linker payload, has superior serum stability while maintaining the CAB target selectivity. Reference: 1. Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, Short JM. Proc Natl Acad Sci U S A. 2021 Mar 2;118(9):e2020606118. 2. Jing Wang, Charles Xing, Haizhen Liu, Ana Paula Cugnetti, Christina Wheeler, Mathew Lucas, Gerhard Frey, Cathy Chang, William J. Boyle, Jay M. Short. Novel conditionally active ADCs (CAB ADCs): A novel class of molecules targeting solid tumors. AACR Annual Meeting 2020, Abstract #7890, Poster #4560

Abstract 1542: Generation of an antibody-drug conjugate-optimized TLR 7/8 agonist payload

Ligation of toll-like receptors 7 and 8 (TLR7/8) can potently activate innate immune cells, including tumor-associated macrophages, to prime downstream T cell activation and drive potent, lasting anti-tumor immunity. TLR7/8 agonists have long been pursued as an anti-cancer therapeutic because of their potential in re-programing the immune system but the clinical utility has been limited by systemic toxicity. Several means of decreasing this systemic toxicity have been investigated, including intra-tumoral administration, nanoparticle encapsulation and conjugation to a tumor-targeting antibody (antibody-drug conjugates, ADCs). ADCs are a clinically validated technology designed to target drugs to disease tissues to reduce the systemic toxicity of highly potent payloads and improve anti-tumor activity. We have developed an imidazoquinoline-based dual TLR7 and TLR8 small molecule agonist that has been specifically designed as an ADC payload. The initial compound was chosen from a set of imidazoquinoline-based small molecules using HEK293 reporter cells expressing either TLR7 or TLR8. This newly identified TLR7/8 agonist potently reactivated immunosuppressive macrophages to produce inflammatory cytokines, increased phagocytosis of tumor cells and enhanced T cell activation and proliferation. A lead payload candidate based on a modified version of the initial TLR7/8 agonist was designed to decrease drug permeability to minimize non-targeted systemic immune activation. The lead TLR7/8 agonist was shown to be significantly less potent than the first-generation compound as a small molecule but demonstrated enhanced immune-stimulating capability when conjugated to a direct immune-targeting antibody. The increased potency as an ADC is hypothesized to be driven by enhanced intracellular retention of the less cell permeable payload and/or a higher binding affinity to the TLR7 or 8 receptors, which was predicted via modeling. The greater in vitro potency of the lead payload was also observed in vivo in an MC38 syngeneic tumor model where 33% complete tumor cures were observed with the lead versus only tumor delay with the permeable version. Drug linkers employing the lead payload were evaluated by varying payload linkage chemistry, drug linker hydrophobicity, and drug release mechanisms using in vivo anti-tumor activity in different syngeneic mouse tumor models as a readout. The optimized drug linker design increased the in vitro and in vivo potency up to 4-fold when compared with the initial linker/payload. These data clearly demonstrate the potency of TLR7/8 agonists as immune stimulants in anti-cancer therapies and outline how their optimization through chemical modification can make them suitable payloads for ADCs.

Abstract 1544: MBK-103, a potent novel conjugation platform-based antibody-drug conjugate, changing therapeutic options in folate receptor alpha positive cancer patients

Folate receptor alpha (FOLR1) is a clinically validated target, that is overexpressed extracellularly in numerous solid malignancies with a high unmet medical need. Ovarian, non-small cell lung and breast adenocarcinomas are among indications with the highest frequency of FOLR1-positive patients with one out of two patients showing upregulated topoisomerase I expression. MBK-103, an antibody-drug conjugate targeting FOLR1, is based on Mablink’s novel proprietary hydrophilic drug-linker platform. It is comprised of: (i) an Fc-attenuated humanized IgG1 monoclonal antibody, that selectively binds to FOLR1; (ii) a polysarcosine hydrophobicity masking entity allowing for a high drug-to-antibody ratio (DAR) of 8, while improving the pharmacokinetics and tolerability of the drug; (iii) a proprietary dipeptide cleavable unit and iv) exatecan, a potent topoisomerase I inhibitor. MBK-103 showed excellent ex vivo stability in human plasma enabling a prolonged half-life, similar to that of the unconjugated monoclonal antibody. This led to an increased drug exposure, resulting in a potent antitumor efficacy observed in a collection of more than ten in vivo mouse tumor models with a diverse FOLR1 expression, already at doses as low as 1-3 mg/kg. In addition, the distinct mode of action of exatecan along with the unique characteristics of the proprietary drug-linker platform resulted in the in vivo antitumor potency obtained also in colorectal cancer models, in which tubulin-inhibiting agents exhibit only limited therapeutic effects. The compound was also very well tolerated in cynomolgus monkeys with HNSTD at 50 mg/kg and even when administered repeatedly. High conjugation yields along with 100% homogeneity are major drivers of the successful ongoing manufacturing process with planned IND submission in Q4/2023.

Abstract 1545: Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins

In healthy cells, the membrane lipid phosphatidylserine (PS) is confined to the inner leaflet of the plasma membrane but in cancer cells and in the tumor vasculature PS is prevalent in the outer leaflet where it is highly immunosuppressive. In this study engineered proteins, such as Bavituximab, a chimeric monoclonal antibody that binds to PS via the abundant serum protein β2-glycoprotein 1, and constructs containing the PS binding domain of β2-glycoprotein 1 (betabodies), are conjugated to anticancer drug-linkers designed to be cleavable by cathepsin B. Cathepsin B is a cysteine protease that is abundant in the lysosomes of normal cells but is upregulated and trafficked to the cell surface and is secreted into the extracellular parenchyma in solid tumors, including breast and pancreatic cancer. A series of drug-linker constructs were synthesized that incorporate unique small-molecule payloads with dual mechanism of action, functioning as both potent tubulin-binding antimitotic agents and vascular disrupting agents. The payloads include benzosuberene-based aniline KGP156 and its phenolic congener KGP18, dihydronaphthalene-based KGP05, and indole-based OXi8006. These drug-linker constructs were reacted with N-acetyl-L-cysteine (NAC) and evaluated for cathepsin B cleavage, and liberation of payload after spontaneous 1,6-fragmentation and decarboxylation. The drug linker construct, NAC-succinimidocaproyl-L-valine-L citrulline-p-aminobenzyl carbamate derivatized with aminobenzosuberene, KGP156, was cleaved by cathepsin B (260 nM) with release of payload at the rate of 12.7 μM/min and by cathepsin L (600 nM) at a rate of 11. 5 μM/min. Other drug-linker constructs in this series demonstrated slower release of payload upon cleavage with cathepsin B or cathepsin L. The drug-linker construct incorporating aminodihydronaphthalene-based KGP05 released KGP05 at a rate of 2.4 μM/min with a comparable rate upon exposure to cathepsin L. Payload release from drug-linker constructs was not observed, over 24 h in the absence of enzyme. Drug-linker constructs without the Val-Cit dipeptide were not cleaved by cathepsin B. The most efficient cathepsin B drug-linker substrate, Mc-Val-Cit-PABC-KGP156, was conjugated to Baxituximab to form the corresponding antibody-drug conjugate (ADC). Baxituximab was first reacted with 2-iminothiolane, a thiolating agent that reacts with accessible lysine residues. Reaction with Ellman’s reagent revealed an -SH/antibody ratio of 3.5. Conjugation of thiolated Bavituximab with Mc-Val-Cit-PABC-KGP156 gave a drug/antibody ratio (DAR) of 3.6/1 after cleavage by cathepsin B and determination of released KGP156 by LC-MS. These studies revealed 1) distinct differences in cleavage rates for drug-linkers, 2) cleavage by cathepsin L, and 3) efficient cathepsin B mediated release of payload from the ADC.

Abstract 1547: The urokinase plasminogen activator receptor-associated protein (uPARAP) is an attractive target for the development of antibody-drug conjugates (ADCs) for treatment of mesenchymal malignancies

Background: Soft tissue sarcoma (STS) is a complex family of rare malignancies with high unmet medical need. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types, which internalizes fragments of collagen and transfers them to the lysosome for degradation. Besides its physiological role, uPARAP is considered a critical modulator of the tumor microenvironment. Here, for the first time, we explored the molecular epidemiology of uPARAP in sarcoma tissue to validate this receptor as target for the development of ADCs in this indication.

Methods: uPARAP expression was assessed in STS-specific tissue microarrays by immunohistochemistry in tissue samples from more than three hundred individual donors. Stainings were assessed by histopathologic scoring and grouped into high, medium and low expressing subgroups.

Results: uPARAP was found to be strongly over-expressed in a high percentage of cases of common STS, with variations in terms of incidence and level of expression between histological subtypes. High uPARAP expression was found in fibro- (86% of cases), synovial (84%), dedifferentiated lipo- (68%), pleomorphic lipo- (65%), myxofibro- (59%), leiomyo- (49%), and myxoid liposarcoma (24%). Work is ongoing in additional sarcoma subtypes.

Conclusions: Based on protein expression uPARAP is an attractive emerging therapeutic target for the development of uPARAP-binding ADCs in a broad range of mesenchymal malignancies. In addition, expression of uPARAP in STS may serve as a potential marker for patient selection in early clinical studies with uPARAP-targeting ADCs.

Abstract 1548: CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells

The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short, and overcoming therapeutic resistance has proven difficult. The presence of chemoresistant leukemic stem cells (LSCs), among other mechanisms, has been implicated as a driver in the observed relapse. Antibody-drug conjugates (ADC) are targeted cancer therapeutics that leverage antigen specificity to deliver a potent cytotoxic payload. Recently, ADCs have shown promise in the clinic, but can also present safety and efficacy concerns. Mylotarg, an FDA approved CD33-directed ADC used to treat Acute Myeloid Leukemia, was the first ADC to be approved. The therapeutic is active against most AML tumors but can lead to severe myelotoxicity given the expression of CD33 on normal myeloid progenitor cells. We leveraged a pharmaco-pheno-multiomic analysis of primary AML tumors to uncover a previously unexplored target expressed on both LSCs and AML blasts, but not normal hematopoetic progenitor cells or other normal tissue. The target also showed capacity to rapidly internalize and has activity of well-established oncogenic kinases associated with expression. A novel monoclonal antibody (IgG1) with favorable binding and biophysical characteristics was established against this novel target and conjugated to a highly potent cytotoxic agent, creating a novel ADC. This ADC shows strong binding across AML tumors with effective internalization and highly potent killing of AML tumors.

Abstract 1549: Preclinical characterization of GQ1010, a next generation Trop2 ADC with the best-in-class potential against diverse Trop2+ solid tumors

Background: While Trop2-targeting antibody-drug conjugates (ADC) have demonstrated meaningful clinical response for the treatment of solid tumors, these ADCs still exhibit significant safety issues that limit the full potential of Trop2 ADCs. GQ1010 was developed based on the intelligent Ligase-Dependent Conjugation (iLDC) technology platform of GeneQuantum to enhance the therapeutic window of Trop2 ADC by utilizing a stable linker and a next generation camptothecin analogue. Here we report that GQ1010 demonstrates superior anti-tumor activities against benchmark Trop2 ADCs while maintaining excellent safety profiles, and a potential for a larger therapeutic window, supporting further clinical investigation of GQ1010 in patient with diverse tumors.

Method: To characterize GQ1010 in vitro, assays for target binding affinity, internalization, cell cycle, apoptosis, bystander killing, and in vitro cytotoxicity were used. A series of xenograft (CDX) models were used for in vivo efficacy evaluation. Ex vivo plasma stability study was performed to determine the linker stability by monitoring drug-antibody ratio (DAR) change and the payload shedding. In addition, in vivo payload distribution study was performed using a xenograft model. Toxicity study was performed in monkey, and clinical observation, body weight, food consumption, clinical pathology (hematology, coagulation, serum chemistry), and pathology were evaluated.

Results: GQ1010 demonstrated more potent in vitro cytotoxicity than DS1062 in diverse Trop2+ cancer cell lines tested. GQ1010 also exhibited superior bystander killing than DS1062. GQ1010 further demonstrated strong in vivo antitumor activities in different CDX models including TNBC, gastric, head and neck (H&N) and pancreatic cancer, and the efficacies were better than DS1062 and Trodelvy®, consistent with the in vitro data. Ex vivo plasma stability data confirmed GQ1010 contains highly stable linker with a minimal DAR reduction and payload shedding, indicating expanded therapeutic window than the benchmark ADCs. Free payload distribution analysis in NCI-N87 model showed high target-specific payload delivery and ~90X payload enrichment in tumor than in plasma, confirming minimal shedding of the payload from GQ1010 during circulation in vivo. In the monkey toxicity study, GQ1010 demonstrated excellent safety without signal of interstitial lung disease (ILD) up to 80 mg/kg dosing.

Conclusion: GQ1010 consistently demonstrate superior linker stability, in vitro and in vivo efficacies than

DS1062 and Trodelvy®, while maintaining excellent safety. These data suggest GQ1010 has the potential as the next generation Trop2 ADC that can address the critical unmet needs for diverse cancer patients.

Abstract 1580: LCN2 is a therapeutic target against inflammatory breast cancer

Breast cancer (BC) is the most common cancer type accounting for 12% of annual cancer cases worldwide. In the United States, 30% of the newly diagnosed cancer cases in woman are BC. Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. Our research team published that LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines and showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. Therefore, to understand the mechanisms involved after LCN2 knockdown, we did a RNA-sequencing using IBC3 cells transfected with LCN2-siRNA or with a negative control siRNA, where we identified 138 dysregulated genes. Genes as SOX18, which correlates to distant metastasis and poor clinical prognosis in cancer, and as LRP4, which correlates to proliferating clinical features by activating the PI3K/AKT pathway were identified. We used the Ingenuity Pathway Analysis software to identify the pathways regulated after LCN2 knockdown and found 25 canonical pathways altered. These results will be further validated by qPCR and Western Blot analysis. Also, after the identification of LCN2 as a plausible target for therapy against IBC, we worked on the preparation of a Herceptin-conjugated liposome loaded with siRNA against LCN2, to improve internalization in HER2+ IBC cells. We developed a three step reaction to obtain trastuzumab DSPE-PEG(2000) maleimide conjugates used to prepare the liposomal formulation. Trastuzumab conjugated liposomes were prepared and characterized using dynamic light scattering to measure their size distribution. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG(2000) trastuzumab and the trastuzumab-liposome, respectively. There is ongoing work to determine the optimal timepoint for internalization of liposomes into IBC cell lines and further treatment with liposomes charged with siRNA LCN2 to treat cell lines and determine the biological effect with the aim of developing a targeted treatment for inflammatory breast cancer.

Abstract 1604: Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2

Interleukin 13 receptor subunit alpha 2 (IL13RA2) is one of the two major receptors for the cytokine interleukin 13 (IL-13). While IL13RA1 has low affinity for IL-13 and it is expressed ubiquitously in humans, IL13RA2 has high binding affinity to IL-13 and its expression in normal tissues is mainly restricted to the testes. IL13RA2 was initially regarded as a decoy receptor because it has a short cytoplasmic tail with no known signaling motifs, and its binding to IL-13 does not lead to activation of the JAK/STAT6 pathway. However, recent studies demonstrated that IL-13-mediated IL13RA2 signaling occurs via STAT6-independent pathways. IL13RA2 is reported to be expressed at a high frequency in glioblastoma multiforme (GBM) as well as in other solid tumors including melanoma, renal cell carcinoma and adrenocortical carcinoma and is correlated with poor prognosis. ADCT-211 is an antibody-drug conjugate composed of HuCl47, a humanized IgG1 antibody directed against human IL13RA2, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG2000 (drug to antibody ratio ~ 1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-211 in human cancer cell lines and xenograft models, to determine its safety, tolerability and pharmacokinetic (PK) in the rat and to measure IL13RA2 expression by immunohistochemistry (IHC) in human tumor specimens. HuCl47 showed specific binding to recombinant IL13RA2, while it did not bind to IL13RA1. In vitro, ADCT-211 had highly potent and targeted cytotoxicity against various IL13RA2-expressing solid cancer cell lines. In vivo, a single dose of ADCT-211 at 10 mg/kg was able to completely eradicate tumors in the subcutaneous (sc) A375 human melanoma xenograft model and resulted in 8/8 tumor-free survivors (TFS) at the end of the study on day 57. In the sc U251 human glioblastoma xenograft model, a single dose of ADCT-211 at 3.75 mg/kg showed potent and durable antitumor activity and it resulted in 5/10 partial responders and 5/10 complete responders, 2 of which were TFS at the end of the study on day 51. In an MTD study in male rats, ADCT-211 was stable and tolerated up to 24 mg/kg single dose, with exposure data being indicative of a linear PK profile with a half-life of 12-19 days. Expression of membranous IL13RA2 was confirmed by IHC in a panel of primary and refractory GBM samples and malignant melanoma, highlighting them as potential indications for the clinical development of ADCT-211. In conclusion, ADCT-211 demonstrated potent and specific in vitro and in vivo anti-tumor activity and it was stable and well tolerated in the rat, warranting further development of ADCT-211 into the clinic in IL13RA2-expressing cancers.

Abstract 1696: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Antibody drug conjugates (ADC) are emerging as paradigm-changing precision therapeutics. Sacituzumab Govitecan (SG), which combines a Topoisomerase I (TOP1) inhibitor payload (SN38) with hRS7, an antibody targeting the tumor-selective antigen TROP2, is proving to be a successful cancer therapeutic for some highly refractory cancers including triple negative breast cancer (TNBC). However, mechanisms and biomarkers of sensitivity and resistance to SG remain poorly understood. Prior clinical trials have identified positive correlations between TROP2 expression and better outcome for metastatic TNBC patients receiving SG. Additionally, patient-derived specimen analyses by our group identified mutations in both TROP2 and TOP1 in post-progression metastatic lesions, suggesting multifaceted resistance mechanisms.

To systematically interrogate ADC sensitivity mechanisms, we utilized genome-wide CRISPR-Cas9 knockout screening to identify novel regulators mediating SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, and endosome trafficking/sorting. The top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Conversely, the very top SG resistance-inducing hit in the genome was PARG, encoding the glycohydrolase that opposes PARP enzymatic activity. Furthermore, the synergistic lethality between TOP1 and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. In addition, to identify SG/ADC specific genes and pathways, we performed a secondary round of CRISPR screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG.

In conclusion, through a systematic approach using CRISPR screening, we identified several novel resistance and sensitizing pathways that are implicated in ADC delivery and target protein turnover. We are currently prioritizing the druggable genes and pathways in preparation for proof-of-concept studies combining ADCs with select targeted therapeutics to achieve greater tumor killing efficacy.

Abstract 1730: A novel chimeric virus-like drug conjugate (VDC) for the potential treatment of HPV-positive tumors

Introduction: Human papillomavirus (HPV) virus-like particles (VLPs) bind to a wide variety of tumor types via modified glycosaminoglycans (GAGs) found on the tumor cell surface. This finding led to the development of the investigational virus-like drug conjugate (VDC) belzupacap sarotalocan (bel-sar, formerly AU-011), an HPV-derived VLP conjugated to a light-activated cytotoxic payload. When activated by near-infrared light, bel-sar induced rapid tumor necrosis resulting in pro-immunogenic cell death, release of tumor neoantigens and long-term anti-tumor immunity in the TC-1 tumor model. When E6 and E7 expressing TC-1 mouse tumors were treated with the VDC, E7-specific T-cells were detected in the absence of provided tumor antigens. A novel chimeric VDC (cVDC) is now in development, in which E6 and E7 are fused to the L2 capsid protein as a means to potentially further enhance the observed anti-tumor response. This cVDC could allow for the targeted cytotoxicity of HPV-positive tumors in addition to the release of supplemental tumor antigens E6 and E7 within the now pro-immunogenic tumor milieu, potentially leading to a long term anti-tumor response.

Methods: The detoxified sequences of E6 and E7 were engineered as one fusion polypeptide on the C-terminus of the L2 minor capsid protein. Both L2/E6/E7 and L2/E7/E6 protein expression vectors were generated to determine if the order of the proteins impacted L2’s ability to co-assemble with L1, the major capsid protein. The plasmids were co-expressed alongside L1 using the mammalian 293TT expression system.

Results: Both the L2/E6/E7 and L2/E7/E7 fusion proteins were expressed and co-assembled with L1 into chimeric VLPs. Fusion protein expression was validated by western blots for L2, E6 and E7, and VLPs were confirmed by electron microscopy.

Conclusions: Preliminary data indicate that chimeric VDCs containing E6 and E7 can successfully be generated using the 293TT mammalian expression system. Studies evaluating the cytotoxicity and E6 and E7 immunogenicity of the cVDC as well as the impact on tumor targeting are underway.

Abstract 1841: TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation

Introduction: Myeloid cells are present in the tumor microenvironment (TME) of most human solid cancers, playing an essential role to influence local immunomodulatory functions. STING signaling in intratumor myeloid cells can enhance interferon (IFN) production leading to enhanced local innate and adaptive immunity, and synergism with other anti-tumor mechanisms. Here, we used a novel iADC, TAK-500, to selectively activate STING in CCR2-expressing cells. This approach enables systemic delivery and favors intratumor accumulation of a STING agonist to potentially achieve anti-tumor activity.

Methods: TAK-500 and mTAK-500, a murine surrogate, were employed to study the immunomodulatory role and anti-tumor effects in preclinical models. CCR2 protein expression was characterized in the TME of >1,000 primary human tumors including non-small cell lung cancer (NSCLC, 2 cohorts, N= 411), colorectal carcinomas (CRC, 2 cohorts, N=350) and pancreatic ductal adenocarcinomas (PDAC, 1 cohort, N=228) represented in tissue microarrays using multiplexed quantitative immunofluorescence (mQIF).

Results: TAK-500 triggered dose-dependent monocyte activation in vitro. mTAK-500 induced robust activation of innate and adaptive immune responses both in vitro and in vivo. In syngeneic mouse models with CCR2 expressing intratumoral myeloid cells, mTAK-500 treatment caused accumulation and activation of CD8+ effector T-cells in the TME resulting in prominent anti-tumor activity and enhanced survival. The baseline levels of CCR2-expressing mMDSCs in mouse models positively associated with anti-tumor response to mTAK-500. mQIF analysis of human tumors confirmed high expression of CCR2 in myeloid cells, lower expression in tumor infiltrating lymphocytes (TILs) and absence in tumor epithelial cells. Using an mQIF panel for simultaneous measurement of DAPI, CK, CCR2, CD11b and CD68, we identified CCR2 protein expression in 94% of NSCLCs, 87% CRCs and 89% PDACs. The levels of CCR2 protein were significantly higher in NSCLC than in CRC and PDAC. In NSCLC, high CCR2 protein in myeloid cells was associated with the presence of activating EGFR/KRAS mutations and increased CD8+ TILs and local PD-L1 expression. In CRC, higher levels of CCR2 in CD68+ macrophages were seen in tumors harboring microsatellite instability than in microsatellite stable carcinomas.

Conclusion: The iADC TAK-500 and mTAK-500 induces CCR2-dependent immune cell activation and anti-tumor effect. CCR2 is highly expressed in intratumor myeloid cells from NSCLC. High CCR2 is associated with enhanced local adaptive immune responses and specific clinical/molecular tumor subsets. TAK-500 is currently being evaluated clinically as a single agent and in combination with pembrolizumab in adults with select locally advanced or metastatic solid tumors (NCT05070247).

Abstract 1854: Cancer treatment utilizing virus-inspired particles for the redirection of immune memory

Cancer resistance is a common clinical outcome following immunotherapy, resulting in a significant unmet need for therapies with alternative and highly differentiated mechanisms of action (MOA). VerImmune has pioneered “Anti-tumor Immune Redirection” (AIR), an approach that repurposes a patient’s pre-existing anti-viral or childhood vaccine immunity towards tumor cells for targeted destruction. Mechanistically, this involves VerImmune’s proprietary platform technology known as AIR-ViPs (Anti-tumor Immune Redirection Virus-inspired Particles). ViPs are based on a modified mouse papillomavirus capsid protein of which 60 copies self-assemble into a 20-30nm T=1 icosahedral structure that are then conjugated on their surface with a CD8+ T cell viral peptide antigen. This peptide conjugated platform specifically targets solid tumors and delivers viral peptide antigens to class I major histocompatibility complex (MHC-1) on the tumor cell surface. This leads to the recognition of the tumor by existing memory viral-specific CD8+ T cells. We show here the three-step MOA of AIR-ViPs. The first step is the specific binding of AIR-ViPs to tumor cells by targeting HSPGs on the tumor cell surface. The binding of ViPs is HSPG-dependent as they do not bind to a HSPG-knockout cell line (PGSA-745). To further assess specificity, we pre-incubated ViPs with varying amounts (100ng/mL-1mg/mL) of heparin which resulted in a dose-dependent decrease in binding where the highest dose of heparin was able to completely block ViP binding to tumor cells. The second step of the MOA is the exogenous loading of the CD8+ T cell-specific peptides onto tumor cell surface MHC class I molecules. To prove this, we demonstrated loading of the SIINFEKL OVA peptide on tumor cells incubated with a SIINFEKL-conjugated AIR-ViP using an antibody that specifically recognizes the SIINFEKL/Kb (MHC-I) complex. We further demonstrate exogenous loading utilizing RMA-S cells that are deficient for MHC-I intracellular processing. The third and final step of the MOA is the redirection of antigen-specific CD8+ T cells to kill tumors following peptide loading. Utilizing an in vitro cytotoxicity assay, which involves co-culturing tumor cells and antigen-specific CD8+ T cells in the presence or absence of AIR-ViPs, specific tumor cell killing was observed and quantified using luciferase which acts as a surrogate for cell viability. Using this assay system and HCMV AIR-VIPs, we showed specific immune redirection of human donor CMV CD8+ T-cell cytotoxicity against several human tumor cell lines incubated with HCMV AIR-VIPs but not with the control unconjugated ViPs. Our results demonstrate the potential of AIR as a tumor antigen-agnostic immunotherapeutic. Additionally, the specificity of ViPs to tumor HSPGs makes AIR potentially applicable across many solid tumors regardless of their origin.

Abstract 1863: Evaluation of BDB101, a first-in-class TLR7/8 dual agonist conjugated to an anti-PD-L1 mAb, in cancer treatment

Background Responses to antiPDL1 checkpoint inhibitors (CPIs) in many tumors are governed by the PDL1 expression on tumor and immune cells in the tumor microenvironment (TME). Increasing PDL1 expression on cells may enhance the response to CPIs. Tolllike receptor (TLR) agonists have shown to induce PDL1 expression in the TME and may enhance the antitumor effects of CPIs. The effective dose of TLR agonist administered to patients is limited by systemic toxicities. To overcome this limitation, one approach is to conjugate the TLR agonist to an antibody. This allows for delivery and release of higher doses of the TLR agonist in the TME while limiting systemic exposure.BDB001 is an intravenously (IV) delivered TLR7/8 dual agonist that activates both the innate and adaptive immune system. In clinical trials, BDB001 has been well tolerated and shown promising antitumor responses in monotherapy and in combination with antiPDL1 therapy (Patel M. et al, SITC 2020 and SITC 2021). To further enhance the antitumor effect and allow for increased concentration of the TLR7/8 dual agonist in the TME, we developed BDB101, a proprietary firstinclass TLR7/8 dual agonist - antiPDL1 monoclonal antibody (mAb) conjugate using our TollLike receptor Agonist Conjugate (TLAC) platform. Here we report on the preclinical and nonhuman primate (NHP) evaluation of BDB101.

Methods BDB101 utilizes a cleavable linker, allowing for the TLR7/8 dual agonist payload to be released in the TME and enabling a bystander effect. BDB101 was assessed for in vitro TLR7/8 dual agonist activity and PDL1 binding, and in vivo antitumor efficacy in immune competent mouse models. The pharmacokinetic and pharmacodynamic effects of BDB101 was evaluated in NHP.

Results The conjugated TLR7/8 dual agonist did not change the PDL1 binding specificity of the antibody, and the uncleaved conjugate did not activate TLR7 or TLR8. In the C26 mouse model, a BDB101 mouse surrogate showed a significantly more robust antitumor effect compared to the TLR7/8 dual agonist or the antiPDL1 mAb alone. In NHP, a single IV infusion of BDB101 showed no significant differences in PK parameters between BDB101 and the total antiPDL1 mAb. BDB101 demonstrated remarkable in vitro stability in human plasma for up to 21 days. In NHP, BDB101 was also very stable with minimal release of free TLR7/8 dual agonist payload. Pharmacodynamic studies in NHP were consistent with PK behavior.

Conclusions BDB101 exhibits robust preclinical efficacy and prolonged stability in NHP. The low rate of free TLR7/8 dual agonist payload released into the systemic circulation from the conjugated form of BDB101, should minimize the risk of systemic TLR7/8 dual agonist exposure, while maintaining the ability to deliver the payload to the TME. These results indicate that BDB101 will likely have a good in vivo therapeutic window and strongly support the clinical development of BDB101.

Abstract 1876: NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of potent payload by counteracting MUC16/CA125 effects

Subsets of tumor-produced proteins, referred to as Humoral Immuno-Oncology (HIO) factors, can bind to IgG1 antibodies and suppress their immune-effector activities. Antibody-drug conjugates (ADCs) targeting tumor cell surface antigens can internalize and kill target cells upon liberation of their cytotoxic payload. Binding of the antibody component of an ADC by a HIO factor may potentially hamper the efficacy of a bound ADC. To assess any suppression of ADC’s activity by HIO factors, we evaluated the efficacy of a HIO-refractory ADC, NAV-001, and a HIO-bound ADC, SS1, both targeting mesothelin. The HIO factor MUC16/CA125 protein, which binds to SS1 ADC, was shown to have a negative effect on its internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of tumor cells regardless of MUC16/CA125 expression in vitro (EC50 ~20 pM) as well as in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor as a payload, demonstrated good stability in vitro and in vivo as well as robust bystander activity against tumor and stromal cells not expressing the target antigen mesothelin, while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenograft models from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed for NAV-001 and warrants NAV-001-PNU’s advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers.

Abstract 1881: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-306 targeting IL-13Rα2 in immuno-oncology

POSTER PRESENTATIONS - PROFFERED ABSTRACTS| APRIL 04 2023

Abstract 1881: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-306 targeting IL-13Rα2 in immuno-oncology

Interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed in various types of cancers including glioma and melanoma and is known to be associated with increased tumor invasiveness and metastases. Here, we present the preclinical efficacy and toxicity results for a novel, anti-IL-13Rα2 ADC-LM-306. In vitro binding activity and internalization of LM-306 and LM-106 (unconjugated antibody of LM-306) were assessed in human IL-13Rα2 overexpressing and endogenous cell lines using flow cytometry. The antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity were assessed using viability assays. Cross-species reactivity of drug was assessed in cells expressing cynomolgus, rat and mouse IL-13Rα2. In vivo A375 CDX model was used to assess the effect of LM-306 anti-tumor activity. The toxicity of LM-306 was evaluated in repeated-dose intravenous dose range-finding (DRF) toxicity and single intravenous maximum tolerated dose (MTD) study in cynomolgus monkeys. Both LM-106 and LM-306 showed concentration dependent binding affinity to IL-13Rα2 in various cell lines (Table 1). In addition, LM-106 also showed a dose dependent internalization and ADCC and LM-306 showed significant cytotoxicity in all cell lines (Table 1). Furthermore, LM-306 showed comparable binding affinity to human IL-13Rα2 to its unconjugated counterpart LM-106 (KD:7.43E-10 vs 1.77E-9). LM-306 was also found to be cross-reactive toward cynomolgus IL-13Rα2. In vivo, LM-306 showed tumor growth inhibition at a dose of 1, 3, 10 mg/kg given intravenously once weekly, indicating its strong anti-tumor activity. The highest non-severely toxic dose of LM-306 was determined to be 9 mg/kg in the DRF study and MTD was determined to be 18mg/kg in MTD study for male monkey. In summary, LM-306 showed high binding affinity to human IL13Rα2 expressing cells with potent cytotoxicity in vitro and significant in vivo inhibition of tumor growth, suggesting its therapeutic benefit in immuno-oncology.

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Abstract 1884: DB-1310, a novel Her3 targeting antibody-drug conjugate, exhibits therapeutic efficacy for solid tumors

Background: HER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Its high expression is associated with poor prognosis in several solid tumors. Many anti-Her3 antibodies have been developed for cancer therapy. To date, only patritumab deruxtecan (U3-1402) showed clinical efficacy in both NSCLC and breast cancer among all Her3-targeting therapies. DB-1310 is a HER3 ADC composed of a novel humanized anti-Her3 immunoglobulin G1 (IgG1) monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor payload via a maleimide tetrapeptide-based cleavable linker with a drug antibody ratio (DAR) of approximately eight. In the present study, we aim to evaluate the efficacy and safety of DB-1310 in preclinical models.

Methods: The binding of DB-1310 to Her3 and other HER families was measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast cancer and lung cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3-positive breast cancer and lung cancer xenograft models. In addition, the efficacy of DB-1310 was compared to that of patritumab deruxtecan in tumor xenograft models. The pharmacokinetics and safety profile were also measured in cynomolgus monkey.

Results: In this study, DB-1310 showed high affinity to both Human and Cyno Her3 without cross reacting with Her2, Her4 or EGFR. DB-1310 binds Her3 using a different epitope from patritumab. DB-1310 has high internalization capability and translocates into lysosomes after internalization. In vitro, DB-1310 exhibited cytotoxicity in a panel of Her3- expression human breast cancer and lung cancer cell lines. It also showed dose-dependent antitumor killing activity in a HER3-positive breast cancer HCC1569 xenograft model and a lung cancer NCI-H441 xenograft model. Tumor regression was observed in a NSCLC patient-derived xenograft model with EGFR mutation under DB-1310 treatment. Moreover, DB-1310 showed stronger tumor growth inhibition than that of patritumab deruxtecan at the same dose. The systemic exposure AUC0-last and Cmax of DB-1310 and total antibody increased dose proportionally from 1 mg/kg to 10 mg/kg with low released payload in serum. The DB-1310 also showed good safety profile with highest non-severely toxic dose (HNSTD) above 30mg/kg.

Conclusions: DB-1310 exhibited its efficacy in tumor models both in vitro and in vivo. These studies suggest that DB-1310 as a novel HER3-targeting ADC showed antitumor efficacy for HER3 positive tumors with good safety profile.

Abstract 1887: Preclinical development of BB-1701, a HER2-tageting eribulin-containing ADC with potent bystander effect and ICD activity

Several HER2-targeting antibody-drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. However, these ADC drugs still face challenges of acquired resistance and/or severe adverse effects associate with their payload toxins. Eribulin, a market approval agent for the treatment of metastatic breast cancer and liposarcoma , is a new choice of ADC payload with distinct mechanism of action (MOA). Here we report preclinical studies of BB-1701, a novel HER2-tageting eribulin-containing ADC. BB-1701 exhibited potent and specific cytotoxicity activities to various types of cancer cells where HER2-exprssing levels vary in large ranges. BB-1701 induced tumor-growth suppression, even regression in in vivo models with a single dose of 1-5 mg/kg dosage. In comparison with other HER2-targeting ADCs with different toxin payloads (DM1, MMAE and Dxd), eribulin-containing ADC demonstrated higher potency to cancer cell lines with low HER2 expressing levels (with lower IC50). BB-1701 also showed effective tumor suppression in models insensitive to T-DM1 or T-Dxd. Mode of action studies revealed significant bystander cytotoxicity of BB-1701 to HER2-low cells when co-cultured with HER2-high cells. Additionally, BB-1701 treatment increased ATP releasing and Calreticulin expression, both were deemed immunogenic cell death (ICD) markers. Lastly, repeat dose pharmacokinetic and toxicology studies in nonhuman primates revealed favorable PK and safety profiles of BB-1701 in the clinical intend dosage route and schedule. In conclusion, these comprehensive preclinical data have strongly supported BB-1701 as a potent antitumor agent to cancers including HER2-low expressing and/or cancers insensitive to other HER2-targeting ADCs. A phase I clinical study of BB-1701 in those patients is ongoing.

Abstract 1987: Delivery of potent cytotoxic camptothecin derivatives to tumors by OncoFAP-based small molecule-drug conjugates

Small Molecule-Drug Conjugates (SMDCs) are anti-cancer pro-drugs composed by a tumor-targeting small organic ligand, a linker structure and a potent cytotoxic payload. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal protein overexpressed in multiple types of solid human malignancies. Nuclear medicine studies validated the applicability of the OncoFAP technology for tumor-targeting applications in a wide variety of tumor types. We have recently reported the development of SMDC products in which OncoFAP is used to deliver Monomethyl Auristatin E (MMAE) to tumors, exploiting linkers that are selectively cleaved by FAP. Here we describe the generation and in vivo characterization of novel OncoFAP-based SMDC products bearing potent camptothecin derivatives acting as topoisomerase I inhibitors. Those drug payloads have been already included in approved ADC products (EnhertuTM and TrodelvyTM). The FAP-cleavable linker technology was directly compared against linker strategies used for approved ADCs. Therapy results in preclinical xenograft models of solid tumors revealed that SMDC products activated by FAP show superior anti-cancer activity and deserve industrial development for the treatment of various human solid tumors.

Abstract 1988: FAP-targeted non-internalising drug conjugates: A comparative evaluation between small molecules, antibodies and peptides

Conventional cancer chemotherapy relies on the use of cytotoxic drugs which are not designed and capable of selective accumulation to neoplastic lesions. The conjugation to tumour-targeting delivery vehicles has been proposed as a strategy to enhance the therapeutic index of potent cytotoxic drugs. To date, fifteen Antibody-Drug Conjugate products have received marketing approval for the treatment of solid and liquid human malignancies. We present an in vivo comparative evaluation of ADCs against products based on small organic delivery vehicles (Small Molecule-Drug Conjugates, or SMDCs) or on tumour-targeting peptides (Peptide-Drug Conjugates, or PDCs). All products are designed to target and to be activated by Fibroblast Activation Protein in the tumour microenvironment. Our results reveal that both targeted ADC and SMDC products mediate potent anti-cancer activity and complete tumour remission in a FAP-positive preclinical murine model of cancer, when administered at the same molar dose. Non-targeted albumin and antibody conjugates included in our comparative studies delayed tumour growth but presented suboptimal anti-cancer efficacy in the same cancer model. Our results indicate that both FAP-targeted ADCs and SMDCs are promising product candidates for the treatment of multiple human malignancies.

Abstract 1997: 2C5 antibody modified dendrimer-based mixed micelles for the treatment of multidrug resistant cancers

Purpose: A major hurdle in chemotherapy is multidrug resistance (MDR) observed in tumors. Downregulation of efflux proteins such as P-glycoprotein (P-gp) using small interfering RNA (siRNA) can be an effective way to treat resistant tumors. In this study, monoclonal antibody 2C5 (mAb 2C5) - PEG7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG2k-DOPE and PEG5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle and encapsulate hydrophobic chemotherapeutic drug in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting.

Methods: G4 PAMAM-PEG2k-DOPE was prepared by conjugating G4 PAMAM with para-nitrophenol (pNP)-PEG2k-DOPE. To prepare 2C5-PEG7k-DOPE, mAb 2C5 was mixed with micelles containing pNP-PEG7k-DOPE and PEG5k-DOPE in sodium citrate buffer at pH 5.0 and later adjusted the pH to 8.0. In vitro evaluation of the targeted and non-targeted micelles were performed in both MDA-MB-231 and SKOV-3TR resistant cell lines. Further a xenograft MDA-MB-231 in vivo mice tumor models were performed.

Results: The 2C5-conjugated mixed dendrimer micelles have a uniform size distribution with a slightly positive zeta potential. They show a stable size and zeta potential for 20 days. The morphology of the 2C5 modified micelles was confirmed using TEM imaging. A higher cellular association was observed in the micelles with the 2C5 antibody targeting using fluorescence imaging (DOPE-FITC tagged micelle) in both the cell lines. At 30 minutes the 2C5 targeted mixed dendrimer micelles showed high cellular uptake and was analyzed using flow cytometry. We further evaluated the P-gp downregulation using western blot and observed a significant downregulation of P-gp levels in the targeted formulation versus the non-targeted formulation. The in vivo tumor growth inhibition study in nude NCG 572 MDA-MB-231 tumor bearing mice showed that the immuno-micelles loaded with siMDR-1 and chemotherapeutics resulted in significantly increased therapeutic efficacy compared to non-modified mixed dendrimer micelles.

Conclusions: We have evaluated the 2C5 antibody-modified mixed dendrimer micelles in different cell models, in addition to the models tested in our previous studies. We have successfully established the stability of the formulation in this study and established a 20-day shelf-life. Cellular association, internalization and P-gp downregulation studies show the superiority of the 2C5 modified dendrimer micelles upon non-targeted preparations or free actives.

Abstract 1998: Characterization of immunoliposomes for HER2-targeted delivery of the dual Rac/Cdc42 inhibitor MBQ-167

Breast cancer is the second leading cause of mortality among women in the US. Among the various subtypes, human epidermal growth factor receptor 2 (HER2) positive breast cancer shows a very aggressive and invasive phenotype. This subtype is characterized by the overexpression of HER2 receptor on the cell surface that upon ligand binding and dimerization, leads to overactive cell signaling that promotes cell proliferation and metastasis. Even though therapies have been developed to treat this type of breast cancer by targeting HER2 and preventing dimerization (eg. Trastuzumab), patients can present with acquired or intrinsic therapy resistance. One of the mechanisms of resistance is the compensation of intracellular signaling from other receptors. This signaling converges on guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42 by exchanging GDP for GTP, thus activating downstream effectors that modulate the actin cytoskeleton to promote cell migration and invasion. Therefore, targeting Rac and Cdc42 activation selectively in HER2 positive breast cancer is a promising strategy for overcoming HER2-targeted therapy resistance. Previously, we characterized the dual Rac/Cdc42 inhibitor MBQ-167 that inhibits Rac and Cdc42 activity with IC50s of 103nM and 78nM, respectively. However, there is a need to develop selective delivery systems that transport MBQ-167 directly into HER2-positive breast cancer cells. Our objective is to deliver MBQ-167 selectively into HER2-positive cells using liposomes coated with Trastuzumab, a clinically used monoclonal antibody that targets HER2. We conjugated Trastuzumab to a lipid linker (DSPE-PEG-Maleimide) by reacting Trastuzumab with 2-iminothiolane (Traut’s reagent) under nitrogenated (low oxygen) conditions and then mixing with the lipid linker overnight. This reaction was characterized by measuring the thiol groups formed after the reaction with Traut’s reagent and after mixing with the lipid, followed by mixing the Trastuzumab-lipid conjugate with liposomes containing MBQ-167. To quantify the amount of MBQ-167 in the liposomes, we determined the excitation/emission parameters of the molecule and measured the concentration of MBQ-167 by fluorescence. We found an increase in thiol groups after the reaction with Traut’s reagent, which decreased after mixing with the lipids, suggesting the formation of the DSPE-PEG-Trastuzumab conjugate. Additionally, we determined the excitation/emission parameters (320nm/430nm), quantified a lower limit of detection (LLOD) at 0.1mM, and calculated an encapsulation efficiency of 97% (530μM). Future studies include testing our formulation in vitro and in vivo in HER2+ breast cancer models.

Abstract 2005: FSHR-mediated targeted delivery of paclitaxel immunoliposomes in receptor overexpressing ovarian cancer cells

The purpose of this study was to explore the Fabʹ fragment of anti-FSHR (Follicle Stimulating Hormone Receptor) antibody as a targeting ligand to graft on the surface of paclitaxel (PTX) liposomes (PLs) for site-specific drug delivery in ovarian cancer (OC) cells. Anti-FSHR antibody Fab’ fragment conjugated PTX encapsulated immunoliposomes (ILs) were prepared, characterized, and evaluated to determine their in vitro efficacy in FSHR receptor positive (Caov3) and negative (SKOV3) OC cells. FSHR expression was determined by confocal microscopy and flow cytometry. The results confirmed that FSHR expression levels of Caov3 were higher and no expression was detected in SKOV3. ILs were prepared by covalent conjugation of Fab’ fragments to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. Immunoreactivity of the prepared ILs was determined against Fabʹ in Caov3 cells using flow cytometry and it was observed that after conjugation, Fabʹ remained intact and also maintained the binding characteristic to the receptor. In MTT assay at the end of 48 h, the IC50 value for ILs was found to be 10.8 and 3.5 folds lower than Taxol® and PLs respectively in Caov3. However, being receptor negative, there was no significant difference noticed for IC50 when PLs and ILs were compared for SKOV3. To prove, whether immune tagging improves cell-specific uptake of ILs in FSHR-expressing cells, cell uptake studies were carried out using confocal microscopy and flow cytometry which showed higher uptake of ILs in Caov3 but not in SKOV3. For assessment of anti-angiogenic activity, a wound scratch assay was performed on Caov3 cells. At an equimolar concentration of treatment given to the cells by PLs and ILs, the % recovery of the wound was 1.3 and 2.3 times lower than Taxol® respectively indicating the superior performance of ILs in inhibiting cell proliferation. The quantitation of cell death after treatment was carried out by flow cytometry after staining with propidium iodide and the highest amount of cell death was seen in ILs group for Caov3 cells. Evaluation of cell cycle in the Caov3 indicated superior results of arrest of cells in G2/M phase. Further, an increase in cell death was observed as indicated by the increase in % of cells in subG1 stage. Performance of PLs was similar in Caov3 and SKOV3 leading to almost around 22% of arrest in G2/M stage, however, for ILs, the number of cells in G2/M was 28% for Caov3 cells at the end of 24 h indicating that the targeted formulation was able to better get internalized in the cells due to receptor-mediated uptake. The results highlight the importance of FSHR as a prominent target for OC therapy and the potential of anti-FSHR Antibody Fab' conjugated nanocarriers as a site-specific delivery system to reduce the limitations associated with OC chemotherapy.

Abstract 2007: Evaluation of the antitumor activity of ca102l, a hyaluronic acid and lenalidomide conjugate, in multiple myeloma

Multiple Myeloma (MM) is a rare and incurable hematological malignancy. Despite emerging treatments, there is still an unmet medical need for novel therapeutics to target the inherent and acquired drug resistance associated with MM. CA102L is a conjugate of hyaluronic acid and an anticancer drug designed to deliver its payload, Lenalidomide (Len), to MM cells via the CD44 receptor. Lenalidomide is used as a front-line therapy in MM; The CD44 receptor is highly expressed in MM and is associated with advanced clinical stages and poor survival. In addition to the enhancement of the therapeutic efficacy of Len, a reduction in off-target toxicity is suggested. The present study was designed to evaluate the efficacy of CA102L in MM and identify its potential mechanism of action. The antitumor efficacy of CA102L was tested (in vitro and in vivo) in two MM cancer models, IM-9 and NCI-H929. Protein expression was used to identify the potential targets of CA102L in MM. Both in in vitro and in vivo investigations, CA102L as a single agent displayed an improvement in potency in MM models. In CB-17 SCID mice bearing either IM-9 or NCI-H929 cells, tumor growth inhibition was significantly higher (P< 0.05) in the CA102L (25 mg/kg) treated group compared to that of Len (25 mg/kg). Further, CD31 and hematoxylineosin (H&E) staining of tumor sections from the IM-9 xenograft mice treated with CA102L resulted in greater antiangiogenic and antiproliferative effects compared to the other groups. Prolonged survival was also observed in CA102L monotherapy in H929 models relative to Len (56% vs 11%). Interestingly, in H929 cell viability studies, CA102L and Len at 400μM showed comparable cytotoxicity with a 60% cell growth inhibition, while superior activity was noted against IM-9-treatments (CA102L 60%, Len 20%). Western blot studies confirmed CRBN as the primary target of CA102L. In summary CA102L as a single agent, exhibited greater efficacy than Len in preclinical MM models, thus representing a promising potential therapeutic strategy for MM. Studies presently are ongoing to further evaluate CA102L’s preclinical profile to advance the candidate into the clinic.

Abstract 2008: Induced macropinocytosis by DNA-PK inhibitor for KRAS mutant cancer targeting of albumin-binding peptide drug conjugate

Although Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) is the most well-known tumor driver gene with the highest mutation rate in several types of cancer, there is no effective mutant KRAS targeting drug yet. Recent studies have reported that macropinocytosis can be increased in cancers with the KRAS mutation. Accordingly, several attempts have been made to deliver therapeutic agents to tumor using the phenomena related to albumin metabolism.To target KRAS mutant cancer cells, we adopted a previously developed albumin-binding caspase-3 cleavable peptide doxorubicin conjugate (MPD1). MPD1 is composed of albumin-binding moiety, caspase-3 substrate (DEVD peptide), and doxorubicin. The albumin-bound MPD1 is delivered into KRAS mutant cancer cells via macropinocytosis and induces apoptosis. Then, the induced caspase-3 cleaves DEVD sequence of MPD1 in the tumor microenvironment and produces free doxorubicin successively exerting in situ amplification and overcoming tumor heterogeneity. In this study, we proposed a strategy for effectively delivering and accumulating MPD1 to tumors by promoting altered albumin uptake of KRAS-mutated tumors. We discovered that DNA-PK inhibitor, one of the anticancer drugs that inhibit DNA damage repairing, selectively increases albumin uptake in tumor cells with KRAS mutation. We found that this is due to activated PI3K signaling in KRAS mutant tumor cells. In particular, this investigation has revealed that DNA-PK inhibitor increased albumin uptake by activating the AMPK in PI3K signaling enhanced cancer. AMP-activated protein kinase (AMPK) is a vital cellular energy sensor, so once AMPK is activated by insufficient nutrition level of cells, it activates albumin uptake for nutrient scavenging to restore low energy status. Therefore, because DNA-PK inhibitor activate AMPK-dependent macropinocytosis, it selectively increases albumin uptake in KRAS mutant cancer, enabling tumor targeting of MPD1. In addition, doxorubicin is accumulated in tumors through the caspase-3 amplification system described above overcoming tumor heterogeneity. We demonstrated that MPD1 and DNA-PK inhibitor combination therapy exerted 100% complete remission in the KRAS mutant TNBC xenograft model, suggesting its potent clinical applicability.In conclusion, our results indicate that DNA-PK inhibitor enhances macropinocytosis in an AMPK-dependent manner. We further demonstrate that DNA-PK inhibitor has a dual role of preventing DNA repair as an anticancer agent and elevating nutrient-scavenging pathway in KRAS mutant cancer as a mean of drug delivery. Thus, the albumin-binding self-amplifying MPD1 synergizes its cytotoxic effects at the tumor site with the DNA-PK inhibitors. We believe that this proposed strategy is a valuable approach to broadening the therapeutic benefit for PI3K-activated cancers including KRAS mutant cancer.

Abstract 2009: Gemcitabine-loaded EGFR aptamer for targeted therapy of lung cancer

Lung cancer is the leading cause of cancer-related death in the world and non-small cell lung cancer (NSCLC) is the most common types of the lung cancer. Epidermal growth factor receptor (EGFR) is an attractive therapeutic target due to its high expression in NSCLC. Gemcitabine (Gem) has shown antitumor activity against many kinds of solid tumors, including NSCLC. Aptamers emerged as promising drug delivery vehicles because of their high binding affinity for the target molecules. The objective of this study was to develop the aptamer-drug conjugates using the EGFR-specific aptamer. EGFR aptamers selected by the SELEX process and gemcitabine was internally incorporated into the EGFR aptamer. In vitro binding affinity of EGFR and Gem-loaded EGFR aptamer was evaluated on EGFR-positive A549, H460, H1299 cell lines, as well as EGFR-negative H522 cells using confocal microscopy. To determine whether the Gem-loaded EGFR aptamer was effectively internalized, Z-stack images were acquired after 1 hr incubation of Cy5-conjugated EGFR aptamer at 37°C. The ex vivo biodistribution of Cy7-conjugated EGFR aptamer was investigated in NSCLC xenograft nude mice. To confirm the antiproliferative effects of Gem-loaded EGFR aptamer, the MTT cell proliferation assay was carried out in NSCLC cells. In the confocal image of EGFR-positive cells, Gem-loaded EGFR aptamers were selectively bound to the cell surface, while no aptamer binding was observed in H522 cells. Z-stack imaging showed that internalized Gem-loaded EGFR aptamer accumulated in EGFR-positive cells. The ex vivo biodistribution data showed that the Gem-loaded EGFR aptamer exhibited higher binding in EGFR-positive A549 tumors than scramble EGFR aptamer. A549 cells showed significant reduction in proliferation following treatment with Gem-loaded EGFR aptamer. Our results indicated that Gem-loaded EGFR aptamer has a potential therapeutic agent in EGFR-positive NSCLC therapy.

Abstract 2012: A novel antibody-drug conjugate platform enabling high drug-to-antibody ratios and greater payload flexibility

Historically, increasing the drug-to-antibody ratio (DAR) in an antibody-drug conjugate (ADC) significantly raises aggregation propensity as a result of the hydrophobicity of the payload. Consequently, DARs are commonly limited to 4, forcing developers to incorporate very high potency payloads to achieve the required efficacy. This, combined with chemical instability and off-target uptake, often results in dose-limiting toxicities and narrow therapeutic indexes.

In this study, we used a novel and flexible multivalent linker technology to construct two ADCs targeting HER-2 with the monoclonal antibody, trastuzumab and interchain cysteine bioconjugation. One ADC incorporated monomethyl auristatin E (MMAE) as the drug payload at average DAR 16 whilst the second incorporated the camptothecin analog, SN38, as the drug payload also at average DAR 16. The ADCs were characterized by liquid chromatography-mass spectrometry (LC-MS), size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and SDS-PAGE and were then tested for efficacy in a range of HER-2 positive cell lines in vitro, and tumor growth inhibition studies using NCI-N87 cells in a mouse xenograft.

Both ADCs remained > 95% monomeric (no aggregation) and demonstrated greater chemical stability in ex-vivo serum stability studies compared to comparator ADC constructs utilizing a clinically validated linker and the same payload release chemistry. In in vitro cell killing studies, the ADCs demonstrated target-specific cell killing with the SN38 average DAR 16 ADC achieving a disproportionate 30-fold increase in IC50 (by ADC concentration) compared to a DAR 8 comparator. Dose dependent tumor growth inhibition was observed in an HER-2 positive NCI-N87 mouse xenograft model with no change in mouse body weight, and good systemic exposure.

These data demonstrate our capability, using a novel multivalent linker technology, to construct ADCs with DARs considerably greater than DAR 8 which are chemically stable, shield payloads from hydrophobic interactions (no aggregation), demonstrate good in vivo exposure profiles and which are highly efficacious in vitro and in vivo.

Higher DARs will permit the incorporation of lower potency and targeted small molecule payloads into ADCs which will result in targeted cancer therapeutics with wider therapeutic indexes through the delivery of greater concentrations of payload to the tumor microenvironment and lower, payload-associated, dose-limiting toxicity.

Abstract 2013: Oxidized anthracycline payloads induce anti-tumor immunogenic cell-death and show linker-dependent tolerability when delivered as ADCs

Novel payloads with orthogonal mechanisms of action (MOAs) to established ADC technologies are important for expanding the utility of antibody-drug conjugates (ADCs) in cancer treatment. Anthracyclines, a class of cytotoxic small molecules that include important clinical chemotherapeutics such as doxorubicin, have long been of interest to the ADC field due to their high potency and unique MOA. Interest in anthracyclines has increased in recent years due to their status as canonical inducers of immunogenic-cell death (ICD), a feature that may lead to more durable tumor regressions and increased synergy with immune checkpoint blockade. In this work, we investigate several drug-linkers based on oxidized derivatives of the highly potent anthracycline analogue PNU-159682. Resultant ADCs are potent and immunologically specific with linker-dependent bystander activity in co-culture cytotoxicity assays displaying heterogeneous antigen (Ag+/Ag-) expression. Rodent in vivo anti-tumor activity and tolerability are shown with significant differences emerging between the toxicity profile of the cleavable and non-cleavable analogues. In vitro, tumor cells treated with anthracycline ADCs showed key hallmarks of ICD including markers of endoplasmic reticulum (ER) stress and the induction of ICD damage-associated molecular patterns (DAMPs) such as HMGB1 and ATP. In vivo mouse xenograft studies show increased tumoral F4/80+ macrophage infiltration by IHC in anthracycline ADC treated animals relative to controls. ADCs delivering monomethyl auristatin E (MMAE) show comparable activity in ICD assays, confirming MMAE as an ICD inducing ADC payload alongside canonical inducers such as anthracyclines. Our results indicate there is a therapeutic window in rodents alongside ICD induction by anthracycline ADCs, positioning anthracyclines as a potentially impactful class of ADC payloads.

Abstract 2160: Quantitative proteomics of antibody-drug conjugates and chemotherapy targets in prostate cancer

Introduction: Prostate cancer therapy involves the use of androgen deprivation therapy, chemotherapy, targeted therapy and immunotherapy. Chemotherapy involves the use of anti-tubulins (docetaxel, cabazitaxel), platinum salts, and topoisomerase inhibitors (TOPO1, TOPO2A). There is no biomarker of chemotherapy that is routinely used. We examined 87 prostate cancer samples using targeted proteomics for biomarkers of response or resistance to chemotherapy agents. Biomarkers of resistance include ERCC1 (Platinum), TUBB3 (taxanes), ALDH1A1 (cyclophosphamide), while response biomarkers include TOPO1 (irinotecan, topotecan), TOPO2A (doxorubicin, epirubicin), and hENT1 (Gemcitabine). We also measured markers for several antibody-drug conjugates targets (Her2, Her3, Trop2) in our clinical proteomics (CLIA) platform and several ADC/CAR-T proteins (PSMA, STEAP1, Nectin4, Claudin 18.2. in our research platform

Methods: Tumor areas from Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from clinical samples of prostate cancer received at our CLIA certified laboratory were microdissected and a quantitative proteomic analysis of 72 biomarkers were conducted using selected reaction monitoring mass spectrometry (SRM-MS).

Discussion:[WL1] Androgen receptor was detected in majority of the samples (83%) with a 19x range of distribution (329 amol/ug - 24063 amol/µg). Majority of prostate cancer samples expressed a range of resistance markers for anti-tubulin inhibitors (TUBB3: 69% detection with a 14x range) indicating that the high expressors is likely to be resistant to docetaxel/cabazitaxel based regimens. ERCC1, a marker for resistance to platinum-based agents, was not detected in 26% of cases, potentially enabling a cisplatin/carboplatin-based regimen in 1/4th of prostate cancer patients. Cyclophosphamide could be effective in 10% of cases where ALDH1A1 was not detected. TOPO1, a marker for irinotecan-based therapy was observed in 94% of cases with a range of ~6x (488 - 2760 amol/µg). Other chemotherapy agents that are not routinely used in prostate cancer was observed in select groups. These include doxorubicin biomarker TOPO2A in 34% of cases with 10x range, gemcitabine biomarker hENT1 in 36% of cases (4x range), temozolomide biomarker MGMT (ND in 12% of cases). ADC biomarker analysis revealed HER2 which was observed in 59% of the cases with range of expression from 301 - 1255 amol/µg including a significant population of low HER2 (84%, <750 amol/µg) cases among the detected samples. HER3 was detected in 33% of cases with a 3x range (178 - 580 amol/µg). EGFR was detected in 94% of samples at levels ranging from 131-1125 amol/µg. Trop2 was quantitated in 93% of the cases and had wide range of 37x (437 -16267 amol/µg). The capacity to multiplex 72 protein biomarkers from 2-3 FFPE sections yields a wealth of actionable information for clinical treatment or patient stratification in clinical trials.

Abstract 2171: A multivariate biomarker predicts sacituzumab govitecan response in solid tumors

Despite the recent clinical success of antibody drug conjugates (ADC) in oncology, predictive biomarkers are lacking, potentially limiting their impact. Herein, we evaluated the ability of candidate biomarkers alone and in combination to predict objective response rates observed in solid tumor patients treated with the TROP2-targeted ADC, sacituzumab govitecan (SG), as determined in the IMMU-12-01 basket trial. We leveraged available next generation sequencing (NGS)-based molecular profiling data from an independent advanced solid tumor cohort (n = 23,968) and developed a multivariate biomarker algorithm that produced biomarker positive rates correlating with the objective response rates (ORR) observed in IMMU-12-01. Candidate biomarkers evaluated included TROP2 gene expression, proliferation (by gene expression) and tumor cellularity. Notably, while TROP2 gene expression was highly correlated with protein expression across 45 tumor types (r = 0.93), TROP2 gene expression alone did not significantly predict ORR across 9 tumor types (r = 0.40, p = 0.29). In contrast, a biomarker algorithm combining TROP2 and proliferation by gene expression with tumor cellularity strongly predicted response both when using tumor type-specific biomarker rates in a discovery cohort (r = 0.83, p = 0.006) and in an independent validation cohort (r = 0.82, p = 0.007). These results indicate that the multivariate biomarker accounts for 67% of the variability observed in response rates and may thus identify patients likely to benefit from SG. Among tumor types with objective responses in IMMU-12-01, biomarker positive rates ranged from 9.9% in colorectal cancer to 57.4% in bladder cancer. Additional tumor types with biomarker positive rates >30% included cancers of the head and neck, cervix, salivary gland, skin (non-melanoma) and ovary, all with positive biomarker rates >30%. Interestingly, most tumor types had biomarker positive rates >5%, suggesting the potential for a tumor type-agnostic approach to patient selection. Considering SG and other ADC’s mechanism of action, a plausible model for response is that (1) higher target expression increases ADC drug delivery, (2) higher tumor cellularity increases ADC bystander effect and (3) higher tumor cell proliferation increases tumor cell death. In summary, we uncovered a novel biomarker algorithm capable of predicting SG response across solid tumors that may be generalizable to ADCs as a class, with the potential to further optimize use and maximize benefit.

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors.

Abstract 2634: AMB302/GR1017, an antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor shows therapeutic potency in orthotopic glioblastoma PDX and bladder cancer models with FGFR3-TACC3 fusion

Background: FGFR3-TACC3 (F3-T3) fusion leads to FGFR3 kinase activation constitutively and acts as a driver mutation in several solid tumors. AMB302/GR1017 is a novel FGFR3 targeting ADC that was developed using the intelligent Ligase-Dependent Conjugation (iLDC) technologies from GeneQuantum (GQ) that provide high homogeneity, excellent druggability and high linker stability, and contains a topoisomerase 1 inhibitor as a payload. Based on the preclinical characterization, AMB302/GR1017 shows impressive anti-tumor activities against glioblastoma (GBM) and bladder cancer (BC) models with either FGFR3 amplification or F3-T3 fusion, and demonstrates the potential as a first-in-class FGFR3 ADC against FGFR3 active solid tumor indications.

Methods: in vitro anti-tumor effects and mechanism of action for AMB302/GR1017 were assessed on patients-derived cells with F3-T3 using a 3D-spheroid high throughput assay. in vivo anti-tumor effect of AMB302/GR1017 were assessed on F3-T3 fusion GBM orthotopic PDX model and several FGFR3+ or F3-T3 fusion BC models

Results: AMB302/GR1017 was generated by linking FGFR3 targeting antibody (AimedBio) with TopoIx (GQ), a next generation Topoisomerase 1 inhibitor via a cleavable linker using the iLDC technologies. In a 3D-spheroid high throughput assay system, AMB302/GR1017 showed significant anti-tumor activity against glioblastoma patients-derived cells (PDCs) in F3-T3 dependent manner, which is superior to chemical conjugate applying the same antibody and Dxd payload. Also, AMB302/GR1017 prolonged the survival of GBM orthotopic PDX models with F3-T3 fusion by 200 % and achieved complete tumor regression in RT112 BC model with F3-T3 fusion. AMB302/GR1017 treatments were well-tolerated up to 200 mg/kg in a mouse safety study.

Conclusion: AMB302/GR1017 showed robust anti-tumor efficacies in F3-T3 fusion and FGFR3 overexpression models derived from GBM and BC in vitro and in vivo. In addition, AMB302/GR1017 was well tolerated with no adverse effects in rodent model. Our data suggest AMB302/GR1017 has a potential therapeutic option as a first-in-class FGFR3 targeting ADC for GBM, BC, and other solid tumors with FGFR3 overexpression or alterations.

Abstract 2635: N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates

Introduction: There is a need for ADC payloads with an improved therapeutic index, consequently we have coupled potent and selective inhibitors of NMT to trastuzumab to test their potential in both in vitro and in vivo models.

Experimental Procedures: A highly potent NMT inhibitor was conjugated to trastuzumab using two different linkers to produce an ADCs with a DAR of 5. These ADCs were tested in vitro in cell killing studies using Her2+ and Her2- cell lines and subsequently tested in vivo in breast cancer and gastric cancer xenografts.

Results: Our first Trastuzumab-NMT inhibitor ADC (MYX2449) caused cell death in Her2+ BT474 cells with an EC50 of 0.2nM and peaking after 8 days of incubation, but was inactive against Her2- cells MCF7 at concentrations up to 100nM. MYX2449 was tested in a BT474 xenograft implanted orthotopically in the mammary gland fat pad and compared to Trastuzumab and vehicle controls. Both antibodies were dosed IV once a week for four weeks at 2.5 and 5 mg/Kg. MYX2449 therapy at 2.5mg/Kg resulted in a partial response (TGI 55% on day 21) and 5mg/Kg resulted in a TGI of 108% on day 21 with 7/10 mice having undetectable tumor by day 33 with no significant body weight loss during the study. Trastuzumab resulted in no response at 2.5mg/Kg a partial response at 5mg/Kg. MYX2449 was subsequently tested in a gastric cancer xenograft, NCI-N87 inoculated subcutaneously and compared to an NMT inhibitor conjugated isotype control and to Trastuzumab and Trastuzumab deruxtecan. All antibodies were dosed IV at 2.5mg/Kg or 5mg/Kg on day 1 and day 8. Day 21 TGI for the 5mg/Kg Trastuzumab group was 193%, for the 5mg/Kg Trastuzumab-deruxtecan group TGI was 154% and for the 5mg/Kg MYX2449 group TGI was 280%. In the MYX2449 5mg/Kg group, 6/10 mice had undetectable tumors on day 21 and 2/10 of the MYX2449 2.5mg/Kg group. Mean body weights of all groups had increased by day 21.

Conclusions: Potent small molecule inhibitors of NMT can be readily conjugated to therapeutic monoclonal antibodies to generate novel highly effective and well tolerated ADC payloads. NMT inhibitors represent a totally novel class of ADC payloads that exploit cancer cell dependency on myristloylated proteins. The potential of this novel mechanism to deliver high efficacy with better TI is currently being investigated in preclinical models. Examples of Myricx patented chemical structures will be disclosed.

RS, ND, FF and RC are employees of Myricx Pharma.JW, FO and ET are funded by Myricx Pharma.ET is a board member and founder of Myricx Pharma.

Abstract 2636: Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Background: Amatoxin-based antibody drug conjugates (so called ATAC®s) constitute a new class of antibody-drug conjugates (ADCs) which utilize amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits cellular transcription. In the current study, in vitro and in vivo data of an ATAC® targeting GCC (guanylyl cyclase C) are presented. GCC is a cell surface receptor expressed in >95% of colorectal cancer, and in approximately 65% of esophageal, gastric, and pancreatic tumors. In healthy conditions GCC expression is restricted to the gastrointestinal tract, and more specific to the apical brush border of the intestinal epithelium (luminal site). Thus, GCC in healthy tissue is not exposed to the circulation but upon tumor progression in GI malignancies it becomes accessible for i.v. injected targeted therapeutics. This tumor-specific accessibility for drugs in circulation makes GCC a highly attractive target for ATAC®s.

Material and Methods: ATAC®s: Cysteine-reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ATAC®s with a DAR of 2.0. Animal models: Subcutaneous (s.c.) mouse xenograft models with the GCC overexpressing cell line HEK293-GUCY2C mono2 or colon cancer PDX models (Charles River) were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in NMRI nude mice and non-human primates (NHP).

Results: All anti-GCC ATAC®s with amanitin-linker derivatives, optimized for use in solid tumors, showed favorable in vitro cytotoxicity with picomolar activity on GCC+ cell lines and no cytotoxic activity on target-negative cells. In mouse xenograft models, the optimized anti-GCC ATAC®s caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Even in colorectal cancer PDX models, anti- GCC ATAC®s led to a substantial anti-tumor effect. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ATAC®s tested in mice differed between 7.5 and 50 mg/kg depending on linker chemistry. Safety profiling in cynomolgus monkeys revealed good tolerability for all selected ATAC®s. Hematology and clinical chemistry parameters were unaffected apart from a moderate and transient increase in liver enzymes.

Conclusions: Targeted cytotoxic drug delivery to GCC+ cell lines was achieved by using anti-GCC ATAC®s optimized for the use in solid tumors. The mode of action of the payload amanitin led to a pronounced anti-tumor effect in vitro and in vivo in PDX models with good tolerability in non-human primates. The use of anti-GCC ATAC®s with their novel mode of action in the therapy of GCC positive cancers, such as colorectal cancer represents a promising approach in cancer therapy.

Abstract 2637: Novel folate receptor alpha-directed antibody-drug conjugate PRO1184 demonstrates broad antitumor activity with a promising safety profile in preclinical models

Folate receptor α (FRα) is overexpressed in a range of solid tumors with limited expression in normal tissues, hence an attractive target for an antibody-drug conjugate (ADC). PRO1184 is comprised of a human monoclonal antibody that selectively binds FRα, a novel cleavable hydrophilic linker, and a topoisomerase 1 inhibitor payload, exatecan. Previous studies established that a) PRO1184 exerts potent antitumor activity in cell-derived xenograft (CDX) models representing ovarian, non-small cell lung, and breast cancer, b) with pharmacokinetics (PK) akin to that of the unconjugated parent antibody in rats, and c) with a preliminary safety profile that was more favorable than a DXd-based ADC in cynomolgus monkeys. Further characterization of the activity and safety of PRO1184 is reported here. PRO1184 displays strong binding affinity, rapid internalization, and potent cytotoxicity with the HCC1954 (breast cancer), JEG-3 (choriocarcinoma), and HEC-1-A (endometrial cancer) cells, in vitro. PRO1184 at a single dose of 5 mg/kg produced marked tumor growth inhibition in CDX models with all three cell lines. In 14 patient-derived xenograft (PDX) models encompassing ovarian, gastric, breast, and non-small cell lung cancer, PRO1184 at a single dose of 5 mg/kg elicited robust antitumor activity in all four tumor types, with ORR and CR accomplished in 10 and 3 out of the 14 models, respectively. Antitumor activity was observed in xenografts with both high and low FRα expression, suggesting that PRO1184 may benefit patients with a wide range of FRα expression. Two DM4-based benchmarking ADCs produced modest to negligible tumor growth inhibition in head-to-head comparisons in the CDX and PDX studies. In a GLP cynomolgus monkey toxicity study for PRO1184, main treatment-related findings were consistent with those associated with exatecan and included reversible gastrointestinal and hematologic toxicities. There were no toxicity findings in lungs, which may translate to a lower risk of interstitial lung disease (ILD) compared to DXd-based ADCs in the clinic. In summary, PRO1184 is a promising development candidate for the treatment of FRα-expressing solid tumors. A first in human phase 1/2 study in patients with advanced solid tumors is currently recruiting (NCT 05579366).

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic.

Abstract 2640: An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo

Background: Antibody-drug conjugate, also known as a targeted chemotherapy, leverages the targeting feature of mAb and the efficient cell-killing ability of cytotoxin to deliver cytotoxic drugs to tumor specifically. Among the FDA approved 15 ADCs and over 100 in clinical trial, the majority contains a single type cytotoxin payload. Combination of chemotherapies or multiple agents are widely therapeutic approach to enhance treatment efficiency, although frequently combo therapies could lead to higher toxicity, long term chemotherapies could result in drug resistance and tumor relapse. To solve above challenge, here we report an innovative dual-payload antibody-drug conjugate (dpADC) with 2 type of different drug payloads with different MoA, the conjugate is constructed based on unique enzymatic site-specific conjugation technology iLDC/iGDC. The dpADC leads to an enhanced immunogenic cell death (ICD) and antitumor response in vitro and in vivo via a synergistic MoA.

Results: A novel Topo1 inhibitor TopoIx and an immune agonist were stably conjugated to an anti-Trop2 mAb in a site-specific manner, conjugate homogeneity and precise DAR were characterized. The dpADC showed a strong cytotoxicity on the proliferation of Trop2-expressing cells, meanwhile bystander killing activity of TopoIx also effectively inhibits growth ofTrop2-negative cells. This dpADC also triggers IP10/IL6 secretion when co-cultured with PBMC/monocyte and Trop2-positive cancer cells, with induction level correlated with Trop 2 expression level. No free payload was released after 96hr incubation at 37 oC in human plasma, demonstrating high linker stability. The dpADC demonstrated a robust antitumor response against diverse Trop2+ tumors in vivo. Its’ combination with anti-mPD-1 resulted in synergistic anti-tumor response. In a tumor rechallenge model, the mice that had cleared of Trop2+ tumors after dpADC treatment were protected against the re-implantation of tumor cells with same or different neoantigen, suggesting the presence of a robust immunological memory.

Conclusion: In summary, this novel dpADC demonstrates potent antitumor activities in vitro and in vivo and leads to an enhanced tumor cell killing ability and a prolonged immunological memory via a synergistic MoA. Based on this site-specific, dual-enzyme orthogonal catalytic conjugation platform, we can easily integrate MoA-varied drugs in one targeting modality to realize a synergistic, durable anti-tumor therapy.

Abstract 2641: ZW191, a novel FRa-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload

Background: Folate Receptor alpha (FRa) is a validated cell surface cancer target that is prevalently expressed in multiple cancers with high unmet need, including ovarian cancer and other gynecological cancers, while exhibiting minimal expression in normal tissues. Due to FRa’s favorable expression profile, multiple antibody-drug conjugates (ADCs) are being explored in this setting. Here we present the preclinical characterization of a new anti-FRa ADC, ZW191. ZW191 is an antibody drug conjugate (ADC) comprised of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor payload, ZD06519, via a maleimidocaproyl (MC) anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker at a drug-to-antibody ratio (DAR) of 8.

Materials and Methods: The novel antibody and drug-linker components of ZW191 were generated, characterized, and optimally integrated. The apparent binding affinity and cellular internalization of the ZW191 antibody, and the intracellular concentration of the released camptothecin payload, ZD06519, were determined in FRa-expressing cells. Additionally, the binding specificity of the ZW191 antibody was determined using a cell microarray technology to test for binding against over 6,000 full length proteins that are individually over-expressed in human cells. Tumor spheroid cancer cell cultures were utilized to determine the cytotoxicity of ZW191 and the ability of ZW191 to penetrate the layers of the three-dimensional (3D) spheroid. The bystander activity of ZW191 was assessed using antigen positive and negative co-culture experiments. The anti-tumor activity of ZW191 was evaluated in a panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) ovarian cancer models spanning a range of FRa expression. ZW191 was evaluated in toxicology and pharmacokinetic (PK) studies performed in rodents and non-human primates (NHP).

Results: The antibody component of ZW191 features a favorable binding profile with strong and exclusive binding to FRa, and drives superior tumor spheroid penetration, cellular internalization, and payload delivery compared to FRa targeted antibodies used in other ADCs. ZW191 demonstrates potent activity in FRa expressing 3D tumor spheroid cultures and effective bystander activity. In a panel of CDX and PDX models representing a range of FRa expression, ZW191 demonstrates compelling anti-tumor activity at exposures that are estimated to be readily achievable in the clinic. ZW191 was tolerated up to 200 mg/kg in a two-dose rat study and at 30 mg/kg in a two-dose NHP study, with favorable PK. The promising efficacy, tolerability, and PK supports the potential of ZW191 as a novel therapeutic agent that may help address unmet need in patients with high and low FRa-expressing cancers.

Abstract 2642: BL-B01D1, a novel EGFR×HER3-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation

EGFR and HER3, members of the human epidermal growth factor receptor (ErbB) family, are targeted in cancer therapy due to their over-expression and pathway dependence in common human epithelial carcinoma tumors. To develop a promising therapeutic anti-tumor agent, we generated BL-B01D1, an EGFR×HER3-targeting ADC, which can bind to EGFR and/or HER3 positive cells and is expected to be superior to anti-EGFR and anti-HER3 ADCs. It is comprised of a bispecific antibody against EGFR/HER3 (SI-B001), a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04), which is a derivative of the alkaloid camptothecin, driving cell cycle arrest at the S phase and subsequent apoptosis. BL-B01D1 achieves a high drug-to-antibody-ratio (DAR=8) with a highly stable linker.

The pharmacological potential of this ADC was evaluated in comparison to its parental single antigen-targeting ADCs in xenograft models composed of either the human colorectal cancer cell line SW620 or pancreatic cancer cell line BxPC3. The tumor inhibition activity of BL-B01D1 was compared with ADCs prepared from each parental anti-EGFR or anti-HER3 mAb conjugated with the same linker and payload. The bispecific ADC, BL-B01D1 exhibited stronger tumor inhibition capacity than the anti-EGFR ADC and the anti-HER3 ADC separately.

The preclinical studies suggest BL-B01D1, as an EGFR×HER3-targeting ADC, might be a promising novel agent with activity toward a broad range of human cancers. The clinical phase I has been progressing and the available data exhibit excellent efficacy but low levels of targeted toxicity in the non-small cell lung cancer (NSCLC) treatment setting. Overall, these data suggest BL-B01D1 has potential to serve as a novel, efficacious therapeutic agent for NSCLC with similar therapeutic impact as DS-8201 has in breast cancer treatment.

Abstract 2643: BL-M07D1, a novel HER2-targeting ADC, demonstrates potent anti-tumor efficacy in preclinical pharmacodynamic models

HER2 is a member of the epidermal growth factor receptor family. Activation of the HER2 signaling pathway has been shown to strongly promote carcinogenesis. It is therapeutically targeted in cancer owing to its overexpression and pathway dependence in a variety of human carcinomas, especially human breast cancers. We created a promising therapeutic anti-tumor agent, BL-M07D1, an anti-HER2-Ed-04 ADC. It is comprised of a humanized anti-HER2 antibody Trastuzumab, a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04), which is a derivative of the alkaloid camptothecin, driving cell cycle arrest at the S phase and subsequent apoptosis. The BL-M07D1 drug-to-antibody-ratio is 8:1 (DAR=8), similar to Trastuzumab Deruxotecan (DS-8201), while possessing a more stable linker.

To evaluate the pharmacological potential of BL-M07D1, xenograft tumor inhibition assays were used to compare BL-M07D1 with the commercialized HER2-targeting ADCs, T-DM1 and DS-8201, which have been approved worldwide for patients with HER2-expressing tumors. Results from the in vivo murine studies show that BL-M07D1 has strong tumor inhibition effects in multiple cell line-derived xenograft (CDX) tumor models. 1) BL-M07D1 exhibited better anti-tumor efficacy than DS-8201 in CDX with low HER2 expression, human epidermal cancer A431 and human non-small cell lung cancer NCI-H1975. Both models are considered be T-DM1-insensitive. 2) BL-M07D1 exhibited better anti-tumor efficacy in comparison to either T-DM1 or DS-8201 in a CDX with JIMT-1, a HER2-positive human breast cancer cell line. 3) BL-M07D1 exhibits potent bystander effects in a heterogeneous xenograft model of HER2-positive and HER2-negative tumor cells composed of NCI-N87 and MDA-MB-468 cells. In this model, BL-M07D1 exhibited stronger tumor inhibition than T-DM1, consistent with bystander effects that are also exhibited by DS-8201.

In conclusion, in vivo studies suggest that BL-M07D1, a novel HER2-targeting ADC, is potentially more efficacious in a broader patient population than T-DM1, and mediate superior anti-tumor efficacy than DS-8201. The clinical phase I is under way and the available data exhibit excellent efficacy in breast cancer therapy with acceptable tolerability.

Abstract 2644: BL-M02D1, a novel Trop2-targeting ADC, demonstrates robust anti-tumor efficacy in preclinical evaluation

Trop2, also known as trophoblast antigen 2, is a transmembrane glycoprotein. It is therapeutically targeted in cancer due to its over-expression in a variety of human carcinomas. To develop a promising therapeutic anti-tumor agent, we generated BL-M02D1, an anti-Trop2-Ed-04 ADC. It is comprised of a novel monoclonal antibody against Trop2 (hu4D3), a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04). The novel Ed-04 is a derivative of the alkaloid camptothecin and mediates cell cycle arrest at the S phase and subsequent apoptosis. BL-M02D1 achieves a high drug-to-antibody-ratio (DAR=8) with a highly stable linker.

The antitumor efficacy of BL-M02D1 was evaluated in comparison to a commercialized Trop2-targeting ADC, IMMU-132, in xenograft tumor models. BL-M02D1 exhibited stronger tumor inhibition capacity than IMMU-132 at lower doses in the gastric cancer cell line NCI-N87, the breast cancer cell line MDA-MB-231, and the non-small cell lung cancer cell line HCC827 xenograft models. BL-M02D1 exhibited potent bystander effects, exemplified by strong tumor inhibition in a heterogeneous xenograft model of Trop2-positive and Trop2-negative tumor cells (A431 and SW620). This characteristic of BL-M02D1 was also compared to IMMU-132. In the heterogeneous Trop2 xenograft model (A431 and SW620), BL-M02D1 exhibited higher tumor inhibition capacity than IMMU-132, indicating that BL-M02D1 possess a more potent bystander effect than IMMU-132.

In summary, these studies suggest BL-M02D1, a novel Trop2-targeting ADC, is potentially more efficacious in the treatment of Trop2-expressing carcinomas than IMMU-132. The clinical phase I has been progressing and the available data exhibit excellent efficacy in breast cancer therapy with manageable toxicity.

Abstract 2648: In vivo efficacy and preclinical safety profile of NN3201, an anti-cKIT antibody-drug conjugate, for treating SCLC and AML

Overexpression and activating mutations of cKIT are associated with tumor aggressiveness and poor prognosis in various cancer patients such as small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), and mastocytoma. Since small molecule inhibitors including imatinib frequently have a limited therapeutic application due to eventual drug resistance and severe adverse events, we developed the cKIT-directed antibody-drug conjugate (ADC), NN3201, for the treatment of wild-type and mutant cKIT-positive cancers. NN3201 is an ADC conjugate of the antimitotic payload MMAE to the fully human 2G4 antibody, directed against cKIT, via the cysteine re-bridging technology ThioBridge® at a drug-to-antibody ratio (DAR) of 4. NN3201 displayed preferable biophysical properties and broadly potent in vitro antitumor activities across various cKIT-positive tumor cell lines. NN3201 exhibited potent in vivo antitumor activities in cKIT-positive tumor cell line xenograft and patient-derived tumor xenograft models of SCLC and AML, representing robust and durable antitumor responses. Furthermore, in a single-dose toxicity study with cynomolgus monkey, no significant or severe clinical toxicity signs were observed in animals dosed at 1, 2, and 3 mg/kg. Even though a decrease in reticulocyte, red cell mass (hemoglobin and red blood cell), neutrophil, and monocyte counts, along with bone marrow recovery and reticulocyte compensatory mechanism, red blood cell and white blood cell recovery were observed from day 14 in the low and medium dose groups and day 21 in the high dose group, dose-dependently. These preclinical results suggest that NN3201 is a potential therapeutic agent for the treatment of cKIT-positive cancers and support the further clinical evaluation in SCLC and AML.

Abstract 2655: Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma

Previously, we have shown that overexpression of Polo-like kinase (PLK)-1 is associated with poor clinical outcomes in patients with aggressive B cell lymphomas. PLK-1 selective inhibitor, Volasertib (BI 6727), has a potent anti-tumor effect in aggressive B cell lymphomas. Unfortunately, Volasertib was withdrawn in Phase III clinical trials due to severe myelosuppression. In this study, we developed a Volasertib antibody-drug conjugate (V-ADC) using -CD19 antibody Inebilizumab to increase the targeting specificity for B-cell lymphoma cells and minimize the side effects of Volasertib. However, we found that V-ADC treatment exhibited little cytotoxic effects, comparing to free Volasertib treatment in Z138 cells, a mantle cell lymphoma (MCL) cell line. We hypothesized that the expression level/density of CD19 on B-lymphoma cells as well as its gene mutations may affect V-ADC binding affinity required for its endocytosis. We, therefore, established Z138 cell lines with overexpression of either wild-type (WT) or mutant (Mut) CD19. The cell viability at 48h post 10nM V-ADC treatment was determined to be 90%, 61%, and 69% in parent, WT, and Mut CD19-overexpressing Z138 cells, respectively. In comparison, the cell viability with 10nM free volasertib treatment ranged from 3-30% in these cell lines. Therefore, other factors, in addition to CD19 expression, may have also played an important role in V-ADC induced cytotoxicity. Upon endocytosis, the V-ADC will enter the endolysosome where cathepsin, a lysosomal enzyme, cleaves the linker between Volasertib and Inebilizumab and releases free Volasertib into the cells. Our previous works have shown that a starvation condition (culture media with 1% FBS) increased lysosomal cathepsin activity, comparing to the culture media with 10% FBS. Herein, we found that upon treatment with 10nM V-ADC, CD19-overexpressing Z138 cells cultured in media with 1% FBS exhibited markedly increased apoptotic cell death comparing to cells in media with 10% FBS (64% vs 41%, respectively). Furthermore, WT and Mut CD19-overexpressing Z138 cells exhibited 2.8- and 2.4-folds of apoptotic cell death compared to control Z138 cells at 48h post 10nM V-ADC treatment with 1% FBS. The cell apoptosis occurred via upregulation of caspase 3, but not caspase 9, with induction of cleavage of poly (ADP-ribose) polymerase (PARP) and reduction of mitochondria membrane potential. In conclusion, starvation and lysosomal cathepsin activation increased V-ADC-induced apoptotic cell death in CD19 overexpression Z138 cell lines. We are actively investigating the in vivo therapeutic effects of V-ADC in patient-derived xenograft (PDX) animal models of MCL and other aggressive B-cell lymphomas.

Abstract 2658: ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload

Background: Glypican-3 (GPC3) is a cell-surface oncofetal glycoprotein frequently expressed in hepatocellular carcinoma (HCC) with minimal presence in normal adult tissues. ZW251 is an antibody-drug conjugate (ADC) targeting human GPC3, composed of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor, ZD06519, via a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker.

Materials and Methods: Extensive functional characterization was performed to assess the mechanism of action and therapeutic potential of the ZW251 ADC. Antibody binding to human and cynomolgus monkey GPC3 was assessed by surface plasmon resonance and flow cytometry. A screen of off-target binding and target specificity was conducted using a membrane proteome array. ZW251 antibody internalization in GPC3-expressing tumor cell lines was assessed by flow cytometry. In vitro ADC cytotoxicity against tumor monolayers and spheroids was assessed in a panel of HCC cell lines. Tumor cell co-culture assays were also performed to assess bystander-mediated cell killing by ZW251. The pharmacokinetic (PK) profile of the ZW251 antibody was assessed in Tg32 mice expressing human FcRn. Anti-tumor activity of ZW251 was investigated in a large panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models representing a range of GPC3 expression.

Results: The ZW251 antibody backbone demonstrated nanomolar binding affinity to both human and cynomolgus monkey GPC3, and strong binding to target-expressing cancer cell lines. Rapid internalization of ZW251 antibody was observed in GPC3-expressing HCC cell lines. ZW251 exhibited potent and target-specific cytotoxicity in a panel of HCC cells cultured either in monolayer or as 3D spheroids. ZW251 showed effective bystander-mediated killing of GPC3 negative cancer cells when in co-culture with GPC3 positive cancer cells. The ZW251 antibody demonstrated a favorable PK profile in Tg32 mice. A single administration of ZW251 resulted in robust tumor growth inhibition of a large panel of CDX and PDX models representing a range of GPC3-expression. Overall, these results support the potential of ZW251 as a novel therapeutic agent against GPC3-bearing cancers.

Abstract 2660: HMBD-501 - a novel Fc engineered, exatecan-based next generation HER3 targeting antibody drug conjugate shows robust efficacy and tolerability in pre-clinical solid tumor models

HER3 is a member of the epidermal growth factor receptor (ERBB) family of tyrosine kinase receptors overexpressed in a broad range of tumors and is associated with disease progression and poor survival. For example, more than 50% of melanoma, cervical, lung, gastric, colorectal, and ovarian cancers show overexpression of HER3, while in breast, pancreatic and prostate cancers the overexpression ranges between 25-40%. In addition, recent studies show that HER3 expression is significantly induced in tumors during metastasis (in colorectal) and during acquired resistance to tyrosine kinase inhibitors (TKI) in lung cancers. This widespread overexpression of HER3 makes it an ideal target for antibody drug conjugates (ADCs). ADCs are a therapeutic modality that harness an antibody’s target specificity to selectively deliver cytotoxic payloads to tumors. Patritumab-Deruxtecan (U3-1402), a HER3-targeting ADC, showed clinical response in a subset of EGFR mutant and TKI resistant lung cancers in a Phase 1 study, albeit with toxicities including severe interstitial lung disease (ILD) and high grade cytopenias. Mechanistic studies show that these ADC-related toxicities are likely driven by Fc- or micropinocytosis-mediated uptake of ADCs by normal cells. We have developed HMBD-501, a novel anti-HER3 ADC with (i) a high-affinity Fab, which can bind to HER3 in the presence or absence of its ligand NRG1, (ii) an engineered Fc-domain to avoid FcgR binding and potential toxicities, and (iii) site-specific conjugation to a next generation cleavable linker and exatecan-based payload. In our preclinical studies, HMBD-501 demonstrates superior inhibition of tumor growth compared to other exatecan-based anti-HER3 ADCs, in a variety of in vitro and in vivo cancer models with a range of HER3 expression. Additionally, the optimized Fc engineering of HMBD-501 shows enhanced tolerability in vivo indicating a potential reduction in Fc mediated toxicities. Finally, the next generation site-specific conjugation-linker-payload technology of HMBD-501 allows optimal plasma stability and pharmacokinetic profile. In conclusion, HMBD-501 represents a next generation HER3 targeting ADC that has a potentially best-in-class efficacy and tolerability profile. Our findings strongly support the development of HMBD-501 as a promising candidate in the treatment of HER3 expressing solid tumors.

Abstract 2700: ORM-6151: A first-in-class CD33-antibody enabled GSPT1 degrader for AML

Targeted protein degraders (TPDs) have expanded the breadth of therapeutic options through both their catalytic mechanism of action and ability to degrade previously “undruggable” target proteins. Prior reports of small-molecule GSPT1 degraders such as CC-90009 in AML demonstrate potent anti-tumor cytotoxicity, but with a potentially narrow therapeutic index. To increase the efficacy vs. tolerability window of TPDs and improve drug delivery, we introduce TPD-Squared (TPD2 TM), a dual-targeted protein degradation approach of combining the catalytic mechanism of targeted protein degradation with the precision of tumor-targeting therapeutic antibodies. We have previously shown in vitro and in vivo efficacy with a HER2-targeted TPD2 conjugate: ORM-5029. Following on that success, we generated conjugates using a CD33-targeting antibody (OR000283) produced by engineering the FAb (H&L) sequences from gemtuzumab onto an IgG1 Fc with N297A variant to inhibit Fc-γR binding. Medicinal chemistry optimization of linker-payloads led to the identification of ORM-6151, which is composed of SMol006, a highly potent GSPT1 degrader conjugated to OR000283 via a novel β-glucuronide releasable linker. ORM-6151 treatment in CD33-expressing cell lines showed picomolar activity with 10-1000-fold greater potency compared to several GSPT1 degrader molecules, including CC-90009 or Mylotarg, and had robust activity in Mylotarg-resistant lines (AML193 and Kasumi6). ORM-6151 also exhibited picomolar potency in in vitro cytotoxicity to primary relapsed/refractory AML patient blasts, with better potency than CC-90009 and Mylotarg. We evaluated ORM-6151 in several in vivo disseminated xenograft models and observed robust efficacy following a single treatment at doses as low as 0.1 mg/kg. Tumor growth inhibition correlated with the degree and duration of GSPT1 depletion and changes in the expression of previously described integrated stress response biomarker genes. In summary, ORM-6151 is a promising, potential therapy for AML and currently in preclinical development as a first-in-class targeted protein degrader therapy with CD33-targeted delivery.

Abstract 2702: GQ1001 is a next generation HER2-targeting ADC with excellent druggability, safety and potency

Background: GQ1001 was developed to overcome the current limitation of existing HER2 ADCs by using the next generation site-specific conjugation and a stable linker technology, coined the intelligent ligase-dependent conjugation (iLDC) technology. iLDC provides a robust platform for the generation of ADC with high homogeneity and excellent stability. Our data show that GQ1001 is highly homogeneous with strong anti-tumor potency, more importantly, markedly enhanced linker stability and reduced off-target toxicity, and points to a safer HER2 ADC with a larger therapeutic window in human.

Methods: In vitro and in vivo anticancer efficacy and the mechanism of action (MOA) of GQ1001 were assessed using several HER2+ cancer cell lines and animal models. Ex vivo linker stability was assessed by incubating GQ1001 with the plasma from different species. Pharmacokinetics in cynomolgus monkeys and safety profiles in rats and monkeys were evaluated.

Results: GQ1001 was generated by conjugating trastuzumab to DM1 via an unique open-ring containing linker and the enzyme-based site-specific conjugation technology that significantly increases the stability of GQ1001. Optimal physiochemical property of GQ1001was demonstrated from the long-term stability study in liquid formulation. GQ1001 demonstrated similar in vitro anti-cancer activities to T-DM1 in HER2+ tumor cells. However, T-DM1 showed significant non-specific cytotoxicity in HER2- cells while GQ1001 didn’t. In animal studies, GQ1001 induced a robust dose-dependent tumor growth inhibition in multiple HER2-positive CDX and PDX models. From the combo treatment studies, GQ1001 showed synergistic anti-tumor response when combined with HER2-targeting tyrosine kinase inhibitors TKIs and chemotherapeutic agents in multiple HER2-positive models, including those resistant to anti-HER2 TKIs and/or mAbs. In ex vivo plasma stability assay, the DM1-shedding ratio of GQ1001 in human plasma was ~1/100th of that of T-DM1. This supreme linker stability was confirmed by the favorable pharmacokinetics in monkey with extremely low free DM1 exposure in circulation at a significantly lower level than T-DM1. The highest non-severely toxic dose HNSTD of GQ1001 in monkey was 45 mg/kg without any signs of peripheral neuropathy and interstitial lung disease, suggesting GQ1001 will demonstrate excellent tolerance in human. Consistent with this preclinical data, GQ1001 demonstrated superior tolerability profile in heavily pretreated HER2-positive advanced solid tumors patients in a global, open labeled multicenter phase Ia trial.

Conclusions: GQ1001 is a unique HER2-targeting ADC that exhibits markedly-enhanced linker stability and safety profiles. Judging from preclinical data, GQ1001 demonstrates the potential to treat HER2-positive cancer patients, alone or in combination, who have progressed on previous HER2-targeting therapeutics with reduced toxicity.

Abstract 2703: Evaluation of the effect of tumor-targeted and immunomodulating nanoparticles on the activity of cytotoxic T cells using CAR T cells as an in vitro model

In solid tumors, there are multiple barriers that prevent anticancer agents and T cells from being able to be delivered into and reach tumor cells. For effective antitumor activity, it is necessary to have direct interactions between cytotoxic T cells and tumor cells. Hyaluronic acid and Iron oxide both have distinctive properties that are advantageous for the development of biocompatible and biodegradable nanoparticle drug delivery systems for cancer. We have developed several targeting and immunomodulatory ligands that can be conjugated to the nanoparticles while carrying chemotherapy or molecular targeted agents for targeted cancer therapy as shown in Table 1. Although the therapeutic efficacy of those targeted nanoparticles has been demonstrated in mouse tumor models, the effect of nanoparticles and their payload drugs on the activities of tumor specific cytotoxic T cells is still unclear. To determine the effect of those nanoparticles on T cell immune responses, CAR T cells provided a well-suited in vitro cellular model. In this study, anti-mesothelin CAR T cells generated from lentiviral CAR T vector transduced T cells were incubated with nanoparticle/drugs and free drugs. The effect of various nanoparticles on CAR T cell viability, and in vitro cytotoxicity on pancreatic cancer cells was evaluated by live/dead, Alamarblue and SRB proliferation Assays. Our results showed that the incubation of CAR T cells with targeted nanoparticles/drugs did not reduce CAR T cells' viability and did not inhibit T cells' activity in killing tumor cells. The ATFmmp14-HANP/SN38 nanoparticle showed an enhanced effect of CAR T cells. Thus, this nanoparticle/drug has the potential for the development of combination therapy with CAR T cells to improve the CAR T cell delivery while synergizing anti-tumor effect by targeted drug delivery, stroma modulation, and tumor cell killing by drug and CAR T cells.

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Abstract 2708: Nano-immunotherapy: efficacy of nanoconjugate QN-247 in a triple negative breast cancer (TNBC) mouse model

Introduction and Purpose: Triple-negative breast cancer (TNBC) accounts for 12% of breast cancers (BC) in the US and 15% worldwide. It lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Compared to other types of BC, it is more aggressive and has higher risks of recurrence. In the metastatic setting, survival outcomes are much worse than other BC subtypes, with a 5-years survival rate of 8-16%. There is an urgent need to develop better treatment options for TNBC patients. We have developed a novel therapeutic, QN-247, by conjugating an anti-proliferative DNA aptamer to gold nanospheres. The aptamer-gold nanosphere construct shows increased half-life and cytotoxic effects relative to the aptamer alone. QN-247 inhibits nucleolin, a key regulatory protein overexpressed in triple-negative breast cancer (TNBC) cells, thereby reducing their proliferation.

Description of Experimental Procedures: A subcutaneous, TNBC model (MDA-MB-231 xenograft) in nude mice was used in this efficacy study. When tumor volumes reached sufficient size (85 mm3), QN-247 test compound, aptamer alone, and vehicle control was administered daily (QD) at 1 mg/kg via intraperitoneal injection for 12 days. Tumor volumes and body weights were measured regularly through day 20. Nine animals were included in each treatment group. Summary of DataA statistically significant reduction in mean tumor volumes of QN-247 treated mice compared to the vehicle control and aptamer alone was observed. Tolerability of the QN-247 treatment was excellent with no evidence of toxicity.

Statement of Conclusions: This study demonstrates that QN-247 exerts a powerful anti-tumor effect. Additional preclinical mouse xenograft tumor models will be treated with QN-247 to study its effect on other cancers.

Abstract 2710: Developing a novel doppel-targeting antibody-drug conjugate (ADC) for solid cancer

The development of anticancer therapeutics has two main objectives: enhancing efficacy and lowering toxicity. Given these goals, an antibody-drug conjugate (ADC), which delivers highly cytotoxic payloads to tumor tissue specifically, is one of the most favorable treatments. ADC is a growing drug delivery technology, with twelve FDA-approved ADCs as of 2022. Most of ADCs in use today target particular molecules for hematologic malignancies. ADCs for solid tumors are relatively in the early stages of development due to the lack of well-defined solid tumor-specific target molecules. To put it another way, the market has an enormous unmet demand for solid cancer-specific ADCs. As seen by recently approved ADCs and ADCs in clinical trials, finding novel targets for solid cancer has drawn a lot of attention in current development trends. Doppel, a prion-like protein, is transiently expressed in the neonatal brain endothelium; in adults, it is exclusively present in testicular cells. In our previous study, we found that Doppel is specifically expressed only in tumor tissues and tumor blood vessels, but not in normal adult tissues; its expression promoted cancerous blood vessel formation. Then, we generated several monoclonal antibodies which target Doppel; the most effective clone was selected. We observed that angiogenesis-related factors were suppressed in the tumor endothelial cells treated with the Doppel-targeting antibody. In addition, we injected the antibody into mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS); the antibody accumulates in the tumor significantly higher compared to non-targeting IgG. We suggest what we believe to be a novel strategy to target a molecular marker that is altered and overexpressed specifically in tumor tissues and tumor endothelial cells. Based on the above discoveries, we have developed the Doppel-targeting ADC; this drug, according to our hypothesis, will target only cancer cells that express Doppel and result in apoptotic cell death. The ADC has demonstrated remarkable anticancer efficacy in both cell and mouse models, although it is still in the early stages of research. We believe that the enhanced specificity for targeting tumors, the high tumor-to-organ ratio, and the bystander killing effect would make this Doppel-targeting ADC a successful “solid cancer-specific” one.

Abstract 2711: Smart encapsulation of a MEK inhibitor into M2-9SLP/mApoE-liposomes for specific GBM targeting

Among the main aberrations occurring in GBM, those in MEK/ERK and PI3K/akt/mTOR pathways predominate and confer GBM Stem-like Cells (GSCs) sustained proliferation and resistance to therapy. A panel of eight patient-derived primary GSCs lines have been screened for their sensitivity to a small kinase MEK inhibitor (MEKi) with AnnexinV/PI staining. Among these, five display a sensitive phenotype with at least 50% reduction on cell viability after 72 hours of treatment. Then, four cell lines, two MEKi-sensitive (ICH001 and ICH003) and two MEKi-resistant (ICH013 and ICH027) were selected for a deeper molecular characterization based on MGMT methylation status, mesenchymal index and main hotspot mutations associated with GBM pathology MEKi incubation on GSC caused a prompt phospho-ERK reduction already after 3 hours. Of note, we report a concomitant activation of AKT and downstream molecules pointing to an ERK-mTOR redundant activity. To this end, we combined MEKi to PI3K/akt/mTOR inhibitor and we observed an increased cell death even in GSCs displaying moderate sensitivity to MEKi as single-agent (MEKi: 90% vs MEKi-PI3K/akt/mTOR inhibitor: 30% cell viability). Then, MEKi ability to cross the Blood Brain Barrier (BBB) and target GBM cells was investigated using a transwell BBB in vitro model. The PI3K/akt/mTOR pathway inhibitor, known from the literature to readily cross the BBB, was included as positive control. Obtained results showed MEKi inability to efficiently cross the BBB, thus limiting its utility as GBM therapy. These results suggest the need for a specific drug delivery strategy in the brain that might be therapeutically effective. Recently our laboratory has provided proof-of-concept of a combination strategy based on radiation and adjuvant drug-loaded liposomes (LPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate BBB crossing. To strengthen therapeutic efficacy and to lower off-target effects, we implemented mApoE-LPs with a matrix metalloproteinases 2 and 9 sensitive lipopeptide (M2-9SLP) that allows controlled payload release only in the tumor microenvironment rich in MMPs. To this end, MEKi was encapsulated into the M2-9SLP/mApoE-LPs, and its capacity to promote cell death was evaluated. M2-9SLP/mApoE-MEKi-LPs caused in all the sensitive cell lines GSCs proliferation inhibition and induction of apoptosis upon 72h in vitro treatment indicating that the encapsulation process did not alter drug efficacy. In conclusion, our in vitro results support MEKi encapsulation into M2P/mApoE-LP as nanotherapeutic strategy that could guarantee specific delivery of MEKi in a MMP2-enriched tumor microenvironment without altering its capacity to inhibit GSC proliferation and survival.

Abstract 2716: Further development of microRNA-34a therapeutics to target castration-resistant prostate cancer

Prostate cancer (PCa) is heterogeneous and typically presents with multiple distinct cancer foci. Androgen receptor signaling inhibitors (ARSIs) are standard-of-care therapies for advanced PCa and can prolong patient survival by months. Treatment-naïve PCa harbors a population of stem-like cells (AR-/lo) functionally defined as PCa stem cells (PCSCs). During cancer progression or upon therapeutic treatment, non-CSCs may undergo cellular plasticity and be reprogrammed to PCSCs. As a result, PCSCs may become the predominant cell population in advanced (Gleason 9/10) and ARSI-resistant tumors, and the “root cause” for drug resistance. However, current ARSI-based treatment regimens primarily target AR+ CRPC cells or clones but largely ignore AR-/lo cells. microRNA-34a (miR-34a), a tumor-suppressive miRNA dysregulated in PCa, is a potent PCSC inhibitor by targeting cellular processes essential for CSC survival and functions. Here we aim to develop novel miR-34a therapeutics to target PCa cell heterogeneity and CRPC. miR-34a expression negatively correlates with the tumor stage in PCa samples. And miR-34a expression is significantly reduced in p53 mutated tumors. We explored two novel miR-34a delivery platforms. The first is packaging-free ligand-mediated conjugate utilizing 2- [3-(1,3-dicarboxy propyl) ureido] pentanedioic acid (DUPA) as a high-affinity binding ligand for prostate-specific membrane antigen (PSMA), which is upregulated in PCa. The second platform uses cationic lipid nanoparticles to package miR-34a (LNP-miR-34a). We show that during PCa progression and upon treatment, PSMA expression is upregulated and continues to be highly expressed in CRPC and PSMA expression level negatively correlates with overall survival in PCa. Moreover, DUPA-conjugate can specifically target PSMA-expressing PCa both in vitro and in vivo. DUPA-miR-34a significantly induces target gene silencing in vitro. On the other hand, LNP show good encapsulation efficiency for miR-34a, LNP-miR-34a significantly repressed cell proliferation by downregulating critical stemness factors in a dose-dependent manner. Meanwhile, we are pursuing chemical modifications of the miR-34a mimics aiming to improve serum stability and future in vivo applications. Both fully modified miR-34a (FM-miR-34a) and partially-modified-miR-34a (PM-miR-34a) exhibit improved serum stability without compromising the functionality. Our data suggest that DUPA-miR-34a represents a promising novel therapeutic for PCSC-enriched CRPC that continue to express PSMA, whereas LNP-miR-34a could be a systemic therapeutic for CRPC treatment in the combinatorial setting. miR-34a, as a direct target and a major mediator of p53 functions, may represent an effective replacement therapeutic in p53-mutated advanced PCa.

Abstract 2722: Synthesis and characterization of novel small molecule drug conjugates with different payloads designed to be released in tumor microenvironment by neutrophil elastase

The development of small molecule drug conjugates (SMDCs) is gaining momentum after the 2018 approval of the small molecule radioconjugate lutetium-177 vipivotide tetraxetan. In contrast to the use of antibodies for tumor homing, small molecule binding ligands may result in a better tumor penetration. Also the potential targets for ADCs are limited to internalizing antigens so the ADC can be trafficked to the lysosomes for release and activation of the payload. We sought new mechanisms for extracellular activation of SMDCs using enzymes, which are present in the tumor microenvironment (TME). For example, neutrophil elastase (NE) belongs to a family of serine proteases that degrades elastin and other extracellular matrix proteins. NE also contributes to cancer progression by enhancing tumor evasion and metastasis. NE expression and neutrophil tumor infiltration have been correlated with metastatic potential and poor prognosis. Previous studies have shown that NE efficiently cleaves and activates camptothecin conjugates by cleaving an ester bond1.

Here we describe the successful use of NE for efficient activation and traceless payload release from several unique and proprietary SMDCs. Binding molecules such as a peptidomimetic αvβ3 ligand were linked via appropriate spacers to substrate tripeptides of NE. The C-terminal end was directly attached to different functional groups at various payloads such as camptothecins (CPT), monomethyl auristatin E, kinesin spindle protein inhibitors, and CDK9 inhibitors. Hindered ester bonds, amide bonds, and sulfoximide bonds were found to be efficiently cleaved by NE; conversely, those linkages were highly stable in culture medium and in circulation. Consequently, respective SMDCs are not or are moderately cytotoxic when incubated alone with cancer cell lines. However, in the presence of NE, cytotoxicity of the SMDCs increases by two orders of magnitude to the low nanomolar or subnanomolar range across several cell lines reaching similar potency as the respective payloads alone. In PK studies in mouse, rat, and dog, the SMDC with a CPT payload, shows a half-life of 3.6, 8.3, and 4.2 h, respectively, and excellent stability in circulation over 24 hr with low exposure levels (<1% of SMDC) of cleaved payload detectable. In an MX1 triple-negative breast cancer xenograft model, tumor regressions were observed when the SMDC was administered in a 2 days on/5 days off schedule.

NE is capable of cleaving sterically hindered ester bonds, amides, and sulfoximides to release a variety of potent payloads from different SMDCs in a traceless manner. These molecules represents a versatile strategy for selective delivery of payloads to TME that does not require the tumor target to internalize.

1Lerchen HG, Stelte-Ludwig B, Kopitz C, et al. Cancers 2022

Abstract 2793: Preclinical pharmacokinetics in cynomolgus monkeys and first in human dose prediction of DB-1303, a HER2-targeting antibody-drug conjugate

Background: DB-1303 is a novel antibody-drug conjugate comprised of trastuzumab biosimilar, enzymatically cleavable peptide-linker, and a proprietary topoisomerase I inhibitor P1003. It is designed to have high plasma stability, low free payload in circulation and wide therapeutic index. Herein, the preclinical pharmacokinetics (PK) characteristics of DB-1303 in cynomolgus monkeys is reported, and human PK exposure in a first in human study was predicted using population PK model with allometric scaling.

Material and Methods: The PK parameters of DB-1303 were obtained from single PK and repeated TK studies in female and male cynomolgus monkeys. Concentrations of DB-1303 ADC, total antibody and released payload P1003 were measured using ELISA or LC-MS/MS. In a PK study, single dose of DB-1303 at 1, 3 and 10 mg/kg were determined in cynomolgus monkeys via IV infusion. In TK studies, cynomolgus monkeys were intravenously administered with DB-1303 at 50, 80 mg/kg once every 3 weeks for 2 repeated doses, or 10, 40, 80 mg/kg once every 3 weeks (Q3W) for 3 repeated doses followed by a 4-week recovery. A population PK model was developed based on the collective PK/TK data in monkeys.

Results: The non-compartment analysis PK parameters of three analytes (DB-1303 ADC, total antibody and released payload P1003) are summarized following single and multiple doses of DB-1303 in cynomolgus monkeys. To predict the human PK for DB-1303, a target mediated drug disposition (TMDD) model was constructed, incorporating binding to serum HER2 and subsequent elimination of the complex into a standard 2-compartmental PK model. The 2-compartment linear parameters were scaled from cynomolgus monkey population PK parameters using allometric scaling exponents of 1 for volumes and 1 for clearance. The binding of DB-1303 to HER2 was incorporated into the model and assumed to be the same for HER2 ECD and transmembrane domain. The PK profiles for DB-1303 in human at the proposed doses in the first in human study are predicted. The safety margins from the monkey HNSTD for each proposed human dose are calculated.

Conclusions: DB-1303 exhibited favorable PK characteristics of DB-1303 in cynomolgus monkeys, and is predicted to have large safety margins at the selected doses in the first in human study. At the time of presentation, the first-in-human phase 1/2 study in patients with advanced solid tumors is in progress (NCT05150691).

Abstract 2923: JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73

Background: STING emerges as a key immune sensor and activated STING pathway plays a key role in innate and adaptive immune response. STING agonist has shown promising potency to stimulate anti-tumor immunity. Considering immune-related toxicity due to systemic administration, as well as difficulty in intra-tumor administration, targeted delivery of STING agonist by iADC is warranted. We developed CD73-STING iADC JAB-X1800, which uses anti-CD73 mAb for targeted delivery of payload JAB-27670, a highly potent non-cyclic dinucleotide STING agonist.

Method: Binding assay and HEK293-STING-luciferase reporter assay were performed to test affinity of JAB-27670. Tumor cells with CD73 expression and THP-1 cells were co-cultured in the presences of iADC JAB-X1800 to assess the IFN-β production. The cell killing activity of iADC JAB-X1800 was evaluated when cancer cells co-cultured with human PBMCs. Furthermore, human and mouse plasma stability of iADC JAB-X1800 was assessed. Finally, the anti-tumor effects of iADC JAB-X1800 alone or in combination with anti-PD-1 were evaluated in animal models.

Results: JAB-27670 is a highly potent STING agonist with sub-nanomolar EC50 in binding and luciferase reporter assays. In CD73-expressing cancer cells and THP-1 co-culture experiments, iADC JAB-X1800 induced IFN-β production with significant improvement compared with the free payload. JAB-X1800 also induced potent killing in vitro. JAB-X1800 is stable in human and mouse plasma. A single dose of JAB-X1800 induced tumor regression in MDA-MB-231 xenograft model and hCD73-MC38 syngeneic model. JAB-X1800 conferred immunologic memory when tumor free mice were re-challenged. JAB-X1800 showed synergistic anti-tumor activity with anti-PD-1 in hCD73-MC38 syngeneic model.

Conclusions: The CD73-targeted STING agonist iADC JAB-X1800 demonstrated high stability in plasma, robust activity, and good tolerability in animal models, supporting clinical development of JAB-X1800 alone and in combination with anti-PD-1 for patients with CD73-expressing tumors.

Abstract 2932: Datopotamab deruxtecan (Dato-DXd) enhances antitumor response to PD-1/PD-L1 inhibitors in TROP2-expressing tumors in mice

Background: Trophoblast cell surface antigen 2 (TROP2) is highly expressed in various epithelial tumors, including non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC), and correlates with poor prognosis. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I (Topo I) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd has shown encouraging antitumor activity and a manageable safety profile in patients with NSCLC and TNBC as part of the first-in-human, Phase 1 study (NCT03401385). To enhance clinical responses, clinical trials evaluating combination therapy of Dato-DXd with PD-1/PD-L1 inhibitors in NSCLC and TNBC are currently ongoing (e.g. NCT04526691 and NCT03742102). Here we report preclinical evidence for supporting increased antitumor activity of Dato-DXd in combination with PD-1/PD-L1 inhibitors using a syngeneic mouse tumor model and its impact on tumor immunity.

Methods: MC38 mouse colon adenocarcinoma cells stably transfected with human TROP2 (hTROP2_MC38) were inoculated subcutaneously in immunocompetent C57BL6 mice or immunodeficient nude mice and the antitumor activity of Dato-DXd with or without mouse PD-1/PD-L1 inhibitors was evaluated. The tumor infiltrating immune cells in hTROP2_MC38 tumors were analyzed by flow cytometry at 3 days and 10 days after dosing. In addition, the impact of CD8+ T cell depletion on the antitumor activity of Dato-DXd against hTROP2_MC38 tumors in C57BL6 mice was evaluated.

Results: Dato-DXd showed stronger antitumor activity against hTROP2_MC38 tumors in immunocompetent C57BL6 mice than in immunodeficient nude mice, which suggests that immune cells play an important role in the mechanism of action (MoA) of Dato-DXd. The combination of Dato-DXd and mouse PD-1/PD-L1 inhibitors enhanced antitumor activity compared to monotherapy against hTROP2_MC38 tumors in C57BL6 mice. Activation of the dendritic cells in hTROP2_MC38 tumors in C57BL6 mice was observed after Dato-DXd monotherapy or combination therapy with mouse PD-1/PD-L1 inhibitors, which led to the increase of tumor infiltrating CD8+ T cells. Involvement of CD8+ T cells in the MoA of Dato-DXd was also supported by the result that CD8+ T cell depletion in C57BL6 mice decreased the antitumor activity of Dato-DXd against hTROP2_MC38 tumors. In addition, activation of tumor infiltrating NK cells and macrophages was observed after Dato-DXd monotherapy or combination therapy, suggesting the potential effect on innate immunity.

Conclusion: These results suggest that Dato-DXd may stimulate tumor immunity and sensitize tumors to PD-1/PD-L1 inhibitors. The combination of Dato-DXd and PD-1/PD-L1 blockers could be a valuable therapy for patients with TROP2-positive tumors.

Abstract 2944: Trastuzumab-deruxtecan (T-Dxd) induces superior macrophage/DC activation, antigen-presentation and anti-tumor adaptive immune responses, which can be augmented by CD47/SIRPA blockade

Background: T-DXd is a new antibody-drug conjugate targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1) in patients with advanced-stage HER2+ breast cancer (BC). T-Dxd consists of the anti-HER2 antibody Trastuzumab conjugated to Dxd (a topoisomerase-I inhibitor). While the HER2-mediated internalization of Dxd leads to direct and bystander tumor cell killing, it is unclear whether T-Dxd retains Fc-mediated induction of immunity and how these responses may interact with Dxd-mediated tumor cell killing. Therefore, in this study we investigated tumor-specific innate and adaptive immune responses elicited upon T-Dxd treatment versus other HER2-targeting agents.

Method: We first compared immune responses of T-Dxd versus T-DM1 or Trastuzumab on macrophage activation after co-culture with HER2+ BC cells. We next utilized JEDI T cells to model tumor-antigen presentation by those macrophages. We also investigated the therapeutic effect of T-Dxd in an endogenous HER2 transgenic mouse model, and measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockades.

Results: We found that T-Dxd retains the capability to engage with FCGRs and promote ADCP of HER2+ BC cells by macrophages, with comparable efficacy to Trastuzumab. Interestingly, T-Dxd mediated ADCP combined with Dxd-mediated immunogenic cell death promoted superior macrophage activation over Trastuzumab and T-DM1, resulting in increased expression of antigen presentation genes, chemokines, and interferon-stimulated genes. In co-culture experiments with JEDI T cells, macrophages that have phagocytosed eGFP-expressing tumor cells after T-Dxd treatment showed superior activation of antigen-specific effector T cells. In vivo, T-Dxd demonstrated exceptional tumor growth inhibition over Trastuzumab, accompanied by increased HER2-specific adaptive T cell responses. Lastly, T-Dxd therapy showed synergistic antitumor activity when combined with immune checkpoint blockade of the CD47/SIRPA axis.

Conclusion: T-Dxd retains antibody-mediated antitumor mechanisms of Trastuzumab such as FCGR activation and ADCP of HER2+ tumor cells. Importantly, this is synergized with T-Dxd induced immunogenic tumor cell death, which results in enhanced macrophage activation upon tumor cell phagocytosis. This result in enhanced tumor antigen presentation and activation of adaptive immunity in vitro and in preclinical mouse models, which may illustrate T-Dxd’s superior and long-lasting clinical efficacy in HER2+ BC patients. Combination therapy of T-Dxd with CD47/SIRPA checkpoint blockade resulted in synergistic therapeutic benefits in our mouse model studies, and further clinical research with this combination is warranted.

Abstract 2947: Preclinical evaluation of a novel B7-H4 targeted antibody-drug conjugate AZD8205 as a single agent and in combination with novel PARP inhibitor and checkpoint blockade

B7-H4 is a cell-surface protein overexpressed in several tumor types and is most prevalent in triple-negative breast (TNBC) cancer (74%), ovarian carcinoma (77%), endometrial carcinoma (94%), and cholangiocarcinoma (89%). In contrast, it has a limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). Here we report and characterize the preclinical efficacy of AZD8205, a novel B7-H4 targeted ADC carrying a topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 at a drug:antibody ratio (DAR) of 8. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. A single IV administration of AZD8205 at 3.5 mg/kg provided robust antitumor activity in patient-derived xenografts from TNBC, ovarian and cholangiocarcinoma tumors, with overall response rates in B7H4-expressing samples of 75%, 64% and 21% respectively. Tumor response was found to be correlated to cell surface target expression and homologous recombination repair deficiency status. To further exploit the DNA damage elicited by the specific delivery of the TOP1i warhead, the combination of AZD8205 with a novel poly-ADP ribose polymerase 1 (PARP1) selective inhibitor, AZD5305, was investigated. Combination of AZD8205 and AZD5305 provided higher antitumor activity than monotherapy, even in PARP inhibition-resistant or low B7-H4 expressing PDX tumors. Finally, cancer cell lines were found to express markers of immunogenic cell death and antigenicity post-AZD8205 treatment in vitro. Therapeutic benefit of combining AZD8205 with checkpoint immunotherapies was then evaluated in syngeneic models, demonstrating enhanced antitumor efficacy of AZD8205 when combined with an anti-PD-L1 antibody. These data demonstrate that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase I/IIa study in patients with advanced solid tumors is currently ongoing (NCT05123482).

Abstract 2967: Preclinical development of a B7-H3-targeting ADC with a novel DNA topoisomerase I inhibitor for solid tumors

Introduction: B7-H3 (CD276) is an immunomodulatory protein of the B7 superfamily molecules, it is overexpressed in a wide range of solid tumors. B7-H3 overexpression often correlates with poor survival and higher tumor grade, it has become an attractive target for the development of novel cancer therapies recently. B7-H3 targeting antibody-drug conjugates (ADCs), such as MGC018 and DS7300a, have shown preliminary clinical efficacy. DB-1311 is a B7-H3 targeting ADC comprised of a humanized B7-H3 antibody conjugated to a novel DNA topoisomerase I inhibitor P1021 via a cleavable linker. We herein report preclinical efficacy and safety data of DB-1311 and its differentiation from DS7300a.

Methods: The binding of anti-B7-H3 to other B7 family members were evaluated via ELISA and SPR. Anti-B7-H3 antibody was conjugated to P1021 with different Drug-Antibody-Ratio (DAR) values. The efficacy of B7-H3 ADCs with different DAR values was tested in vivo, pharmacokinetics and safety profile were evaluated in cynomolgus monkeys. Based on potency and toxicity analysis, DB-1311, an ADC with DAR6, was selected for preclinical development. In vitro and in vivo studies were then conducted with DB-1311 to further investigate its pharmacological effects.

Results: B7-H3 antibody with DAR6 demonstrated higher efficacy than its DAR4 counterpart in lung cancer (Calu6), glioma(U87MG) and prostate cancer (PC-3) models. In monkeys, the DAR6 ADC showed good stability and dose-proportionally increased exposure. Additionally, it was well tolerated with repeat dose administration up to 80 mg/kg and didn’t show significantly worse toxicities than DAR4 counterpart. In preclinical study, DB-1311 demonstrated specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro and potent antitumor activity in vivo but minimum tumor growth inhibition activity in a B7-H3 negative xenograft model. Moreover, DB-1311 showed more potent antitumor activity than DS7300a analogue in vitro and in vivo.

Conclusions: DB-1311, a preclinical B7-H3 ADC candidate, with a DAR value of approximately 6, exhibited potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types and was well tolerated in cynomolgus monkeys with promising pharmacokinetics characteristics. The preclinical antitumor activity and safety profile warrants further development of DB-1311 to evaluate its efficacy and safety in clinical studies.

Abstract 2977: A first-in-class anti-HER2/TROP2 bispecific antibody-drug conjugate (YH012) exhibits potent anti-tumor efficacy

Antibody-drug conjugates (ADC) are a therapeutic modality that combines the advantages of potent killing from small molecule cytotoxic payload and highly specific targeting from monoclonal antibodies. By targeting dual tumor-associated antigens (TAA), the bispecific ADC (BsADC) serves as a promising therapeutic strategy that can further increase tissue specificity and selectivity. HER2 and TROP2 are two TAAs that are commonly expressed and co-expressed by multiple tumor types, including gastric, colorectal, bladder, breast, and non-small-cell lung cancer (NSCLC). Of note, TROP2 expression has been detected in a wide range of HER2-low expressing tumors. These data led us to predict that targeting HER2 and TROP2 with a BsADC would provide therapeutic benefit, especially for patients with HER2-low cancers. Hence, we generated YH012, a first-in-class BsADC that contains a fully human bispecific anti-HER2/TROP2 antibody conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker. In vitro, YH012 demonstrated enhanced affinity, internalization, and tumor selectivity compared to its parental monoclonal and monovalent anti-HER2 or anti-TROP2 antibodies. In vivo, YH012 showed strong anti-tumor activity in multiple cell line-derived and patient-derived xenografts (CDX and PDX) of NSCLC, gastric, pancreatic, and breast cancer. Moreover, YH012 exhibited superior anti-tumor efficacy than benchmark antibodies in both HER2-positive and HER2-low xenograft models, indicating that YH012 has a potent and broad therapeutic effect. In summary, YH012 has the advantages of increased potency, tissue specificity, and reduced toxicity as a novel BsADC that can be further exploited to treat HER2 and TROP2 co-expressing tumors, especially HER2-low tumors.

Abstract 2978: YH013, a novel EGFR x MET bispecific antibody-drug conjugate, exhibits potent antitumor efficacy

Bispecific antibodies (BsAb) that target dual tumor-associated antigens have the advantages of invoking synergistic effects between two signaling pathways, increasing target tissue specificity, and reducing systemic toxicity. By combining the antibody-mediated specific targeting with potent killing from a cytotoxic payload, antibody-drug conjugates (ADC), and especially bispecific ADCs (BsADC), have become powerful therapeutic strategies. EGFR and MET are oncogenic proteins that are co-expressed in a wide range of tumors. Moreover, MET amplification is largely associated with drug resistance of EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients. Biocytogen developed a fully human EGFR x MET BsADC using our proprietary common light chain RenLiteTM mouse platform and knobs-into-holes technology. The BsAb showed enhanced internalization and binding affinity compared to parental monoclonal and monovalent antibodies in the EGFR/MET co-expressing NCI-H1975 cell line. After conjugating the BsAb with monomethyl auristatin E (MMAE) via a protease-cleavable linker, the resulting BsADC, YH013, exhibited a remarkable and dose-dependent anti-tumor efficacy in NCI-H1975 and NCI-H292 cell line-derived xenograft models. Moreover, in multiple patient-derived xenografts of NSCLC and pancreatic ductal adenocarcinoma (PDAC), which co-express EGFR and MET, YH013 demonstrated superior and durable efficacy that outperformed benchmark antibodies at a lower dose (3 mg/kg). Collectively, these results suggest that YH013 can be an effective treatment option for EGFR and MET co-expressing tumors and overcome MET-driven EGFR-TKI resistance to improve patient outcomes.

Abstract 2981: Anti-tumor activity of vobramitamab duocarmazine (MGC018), an investigational duocarmycin-based anti-B7-H3 antibody-drug conjugate (ADC), in preclinical neuroblastoma models

Introduction: MGC018 is an ADC composed of a humanized anti-B7-H3 IgG1 monoclonal antibody conjugated via a cleavable linker to vc-seco-duocarmycin-hydroxybenzamide azaindole (DUBA), a synthetic DNA alkylating agent whose cytotoxic activity is cell-cycle independent and retained in multidrug-resistant cells. MGC018 has shown preliminary clinical activity in B7-H3-expressing tumors, including castration-resistant metastatic prostate cancer. Owing to its expression in several childhood tumors, B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB).

Methods: B7-H3 expression was evaluated by flow-cytometry (FCM) in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by MTS assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by the Annexin V staining. Pseudometastatic, orthotopic, and resected mouse NB models were developed via tumor cells tail vein injection, implantation in the adrenal gland, or implantation followed by surgical resection of the primary tumor mass, respectively, to mimic disease conditions related to circulating tumor cells and metastases, primary tumor growth, and minimal residual disease.

Results: All cell lines expressed cell surface B7-H3 in a unimodal fashion, ranging between 200 and 400 in mean ratio fluorescence intensity over unstained cells. MGC018 was cytotoxic in a dose- and time-dependent manner against all NB cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). MGC018 was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of MGC018 when co-cultured with human B7-H3-expressing cells, demonstrating by-stander activity. In the pseudometastatic model and in five orthotopic NB mouse models, weekly iv treatments with 1 mg/kg MGC018 for 3 weeks resulted in delayed tumor growth and increased survival rates compared to animals treated with an irrelevant (anti-CD20) duocarmycin-ADC or an anti-GD2 antibody (dinutuximab beta). A 4-week course of treatment further ameliorated MGC018 antitumor effect in both the orthotopic and resected NB models and increased the survival of NB-bearing mice co-treated with TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for relapsed disease. In the orthotopic model, tumor relapse was temporarily or completely arrested by 2 or 3 courses of MGC018, respectively. MGC018 treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities.

Conclusion: MGC018 exerts relevant antitumor activity in pre-clinical NB models and may represent a potential candidate for future NB clinical translation.

Abstract 3067: Constitutive macropinocytosis in BRAFV600E thyroid cancer promotes response to a serum-albumin conjugate of monomethyl auristatin E

Background: The non-specific uptake of extracellular materials through macropinocytosis can fuel tumor growth and survival, and is especially studied in cancer-types that frequently harbor RAS or PTEN mutations. However, relatively little data describe macropinocytosis in follicular, papillary, and anaplastic thyroid cancers (FTC, PTC, and ATC, respectively) despite the abundance of prominent MAPK and PI3K pathway mutations in these malignancies. We hypothesized that thyroid cancers exhibit elevated macropinocytosis and efficiently accumulate chemotherapy covalently conjugated to serum albumin, a common macropinocytosis substrate.

Methods: We assessed the impact of ectopic BRAFV600E expression or targeted kinase inhibition on macropinocytosis in vitro across a panel of thyroid cancer cell lines with varied BRAF mutation status. Cellular uptake of dextran and serum albumin was quantified across ≥ 246 individual cells using fluorescence microscopy in triplicate experiments. The in vivo uptake of serum albumin by both ATC cells and tumor infiltrating immune cells, including tumor-associated macrophages, was assessed across n = 4 replicates of an immunocompetent orthotopic model of BrafV600E p53-/- ATC in B6129SF1/J mice. The same mouse model was used to measure the effects of an albumin-drug conjugate based on the microtubule-destabilizing monomethyl auristatin E (MMAE) linked to serum albumin via a cathepsin-cleavable peptide (Alb-vc-MMAE) on tumor volume using n ≥ 10 tumors per group. Groups were compared by two-tailed Wilcoxon rank-sum tests.

Results: FTC and ATC cells exhibited greater macropinocytosis than PTC and immortalized follicular thyroid cells. Fluorescence microscopy of thyroid cancer cells revealed punctate subcellular co-localization between dextran and serum albumin. Treatment with the Na+/H+ exchanger (NHE) inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced cellular uptake by roughly 50%, consistent with macropinocytosis (P < 0.01). Ectopic BRAFV600E enhanced macropinocytosis by 2-fold, while BRAF and MEK kinase inhibition decreased uptake by 50% (P < 0.01). In vivo, ATC tumors accumulated serum albumin at 8.5%ID/g (% injected dose per gram tissue). The albumin-drug-conjugate Alb-vc-MMAE, but not MMAE alone, blocked more than 90% of ATC tumor growth (P < 0.01).

Conclusions: ATC exhibits high constitutive macropinocytosis driven by elevated oncogenic signaling, including via MAPK/ERK activity stimulated by BRAFV600E mutation. Macropinocytosis in ATC, therefore, may be a target for manipulating tumor metabolism and has implications for the delivery of some albumin-bound and other drugs that are affected by macropinocytic cell uptake.

Abstract 3242: Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy

Antibody-drug conjugates (ADCs) are effective anti-tumor therapies with high specificity, with little toxicity and side effects. Xenograft tumor models based on immunodeficient mice have been used for evaluating the human anti-tumor efficacy of ADCs. However, the antibody-mediated immune response of ADCs is not well evaluated in immunodeficient mice. To this end, we have developed BALB/c-hPD1 humanized mouse model to evaluate the efficacy of ADCs in combination with immunotherapy.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is a HER2-targeting ADC. In vitro efficacy studies showed that T-DXd effectively bound to the HER2-positive cell line SK-BR-3, resulting in strong endocytosis and induction of apoptosis. It has also been shown to kill HER2-negative MDA-MB-468 cells through a bystander effect both in vitro and in vivo. In vivo efficacy studies have demonstrated that ADCs can effectively inhibit human tumor cell growth in immunodeficient mice. In addition to human tumor cell lines, the efficacy of ADCs was also studied in BALB/c-hPD1mice engrafted with murine colon cancer cells model, and the results showed that T-DXd and Keytruda could significantly inhibit CT26-hHER2 tumor growth, with the tumor growth inhibition rates being 57% and 66%, respectively, Tumor growth inhibition rate of the combined treatment group was about 91%, which was superior compared to the efficacy of single drug treatment. The evaluation of Fc segment-mediated effect (such as ADCC, CDC and ADCP) of ADCs antibodies was carried out in humanized immune system mouse models, such as NCG-hIL15 and NCG-hSGM3. In summary, we have established a drug evaluation platform using various humanized mouse models to study the synergistic anti-tumor effects of ADCs combined with immune checkpoint blockade antibodies.

Abstract 3441: A multivalent peptoid conjugate that inhibits therapy-resistant prostate cancer cell proliferation by modulating androgen receptor transcriptional activity

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance despite the continued expression and function of the AR. We developed a new approach to antagonize the proliferative activity of AR and inhibit castration-resistant prostate cancer (CRPC) growth using multivalent peptoid conjugates (MPCs). Our strategy is to display multiple copies of the AR antagonist, ethisterone, on a peptoid scaffold. Such a multivalent display increases the ligand’s effective local concentration, thus targeting AR with high affinity and affecting AR interactions with coregulators. Here, we report a new potent MPC called MPC309, which displays three ethisterone groups and binds to AR with nanomolar affinity. MPC309 reduces the proliferation of enzalutamide-resistant prostate cancer cells, including those harboring AR splice variants, AR ligand binding mutations, and non-canonical AR gene expression programs. We provide evidence that MPC309 enters cells via macropinocytosis, which facilitates the fluid-phase uptake of extracellular macromolecules. Macropinocytotic uptake of MPC309 enhances cancer cell-specific delivery, thus limiting systemic toxicities. MPC309 displays favorable pharmacological properties and produced significantly greater tumor suppression in xenograft studies than enzalutamide, the standard of care anti-androgen in clinical use. Mechanistically, MPC309 inhibits prostate cancer growth by eliciting a unique gene expression program through alterations in AR chromatin occupancy compared to dihydrotestosterone (DHT) or enzalutamide. Thus, MPC309 represents a novel AR antagonist that activates an AR anti-proliferative program to repress the growth of CRPC and supports further investigation of the MPC compound class for treating CRPC.

Abstract 3782: Targeting cancer cells using folate conjugated to a fully modified version of miR-34a (FolamiR-34a) to produce enhanced and sustained anti-tumor activity

Due to the pleiotropic nature of miR-34a, including its ability to downregulate multiple genes that drive resistance to currently used anti-cancer therapies, we hypothesize that the best use of miR-34a in a clinical setting would be in combination with standard-of-care treatments. For in vivo use, such a molecule would necessitate the following: i) a stabilized version of miR-34a that can withstand the harsh environment in circulation and intracellular nucleases, and ii) a robust, specific, and safe delivery vehicle. With regard to stability, we successfully generated the first fully modified version of miR-34a. This new chemically modified molecule is over 400-fold more stable than the previously used partially modified version and induces enhanced and sustained target gene repression. To assess in vivo activity of fully modified miR-34a requires a suitable delivery vehicle. Indeed, while various approaches have been used to restore miR-34a in tumors, clinically relevant mechanisms for delivery of miR-34a have been a challenge due to poor tumor uptake, unfavorable bioavailability, and unwanted toxicity. Previously, we determined that a partially modified version of miR-34a can be delivered specifically and robustly to cancer cells in a targeted, vehicle-free manner through direct conjugation to folate, a ligand of the high-affinity folate receptors. Thus, we conjugated our fully modified miR-34a (FM-miR-34a) to folate generating FM-FolamiR-34a. Following in vivo delivery of FM-FolamiR-34a to mice with breast cancer xenografts we observed enhanced and sustained target gene repression, in comparison to our first-generation partially modified folate-miR-34a conjugate (FolamiR). Critical miR-34a target genes involved in promoting resistance, including MET, AXL, and CD44 were downregulated over 90% for at least 120 hours following a single 1.5 nmol dose of FM-FolamiR-34a. Our final objective is to evaluate FM-FolamiR-34a in combination with standard-of-care agents in vivo.

Abstract 3942: Differential expression of a novel transport receptor, SORT1 (sortilin), in cancer versus healthy tissues that can be utilized for targeted delivery of anti-cancer drugs

Sortilin (SORT1), or neurotensin receptor-3, is a scavenging receptor in the Vacuolar Protein Sorting 10 protein (Vps10p) family. SORT1 is involved in the internalization and trafficking of its ligands through an endocytic process and is associated with cancer cell survival and progression, making SORT1 a candidate for novel drug delivery. We recently reported on the pattern and prevalence of SORT1 expression in endometrial, breast, ovarian, colorectal, pancreas cancers, and skin melanoma. To better understand SORT1 expression, we screened tissues from different cancer types using the same immunohistochemistry (IHC) method. A total of 19 cancer tissue microarrays (TMAs) with 1394 evaluable cancer cores were screened. Each cancer core was scored using an H-score ranging from 0 to 300, where 0 corresponds to no cell stained for SORT1, while 300 corresponds to strong SORT1 staining in all cells. The table below summarizes the % of cores with moderate to high SORT1 expression (defined as H-score ≥100) as well as the average H-Score for each cancer type evaluated. Sub-analyses of SORT1 expression by tumor histological sub-type, stage and grade are also being performed.

A total of 234 healthy or normal adjacent tissues cores were also assessed. Weak or null staining was observed in these tissues. Moderate staining was observed in specific cell types in kidney tubules and glomeruli, colonic mucosa, splenic sinusoidal spaces in red pulp, blood vessels in smooth muscle of spleen and colon, dendritic and axonal extensions of pyramidal-type neurons in brain, and testicular seminiferous tubules. SORT1 is currently being studied as a cancer target in a first-in-human (FIH) study of a peptide-drug conjugate (clinicaltrial.gov: NCT04706962). These results suggest that SORT1 is highly expressed in multiple tumors and is a promising target for the delivery and internalization of cancer therapeutic agents.

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