AACR 2023 XDC合集（二）

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。

Abstract 3995: Trastuzumab deruxtecan, antibody-drug conjugate targeting HER2, effectively inhibits growth of patient-derived xenograft model with CIC-rearranged sarcoma

Background: Capicua-double homeobox 4 (CIC-DUX4)-rearranged sarcomas (CDS) are extremely rare and highly aggressive sarcomas. There is no standard therapy for the patients with advanced CDS, and therapeutic development is needed. We evaluated preclinical efficacy of trastuzumab deruxtecan (T-DXd), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient-derived xenograft (PDX) models with CDS.

Methods: Patient-derived tumor tissue was transplanted into subcutaneous around the flank of female NOG mice, which were treated with vehicle or T-DXd (3 mg/kg, intravenous, Day0) when the mean tumor volume reached 200 mm3. Tumor volume was assessed twice weekly for 3 weeks to assess the efficacy of T-DXd.

Results: This study included 3 CDS-derived PDXs. HER2 expression was low in one PDX and not expressed in two PDXs. One PDX was established from a specimen at initial diagnosis, two were established from specimens after prior-chemotherapy. T-DXd demonstrated significant tumor growth delay compared to vehicle in all PDX models investigated. One PDX with no efficacy was HER2-negative and had a treatment history of topoisomerase I inhibitor. In contrast, PDX of HER2-negative topoisomerase I inhibitor-resistant Ewing sarcoma was also administered T-DXd under the same conditions and was found to be refractory.

Conclusion: The present study showed that a therapeutic potential of T-DXd in CDS patients, including HER2-negative.

Abstract 3996: The impact of HER3 dynamics on the efficacy of HER3-DXd, a novel HER3 directed antibody-drug conjugate

Background: HER3 is broadly expressed in various solid tumor types, and its expression can be upregulated by treatment with receptor tyrosine kinase inhibitors (RTKi) such as EGFR TKIs used to treat EGFR-mutated NSCLC. HER3-DXd, a novel antibody-drug conjugate (ADC) composed of a human anti-HER3 IgG1 monoclonal antibody (patritumab) covalently linked to a topoisomerase I inhibitor payload (DXd), is currently being studied in clinical trials for breast cancer and NSCLC. As previously reported, HER3-DXd treatment transiently decreases HER3 expression levels in tumors and EGFR TKIs increase HER3 membrane expression. However, the impact of HER3 dynamics on payload delivery has not been clarified yet. In this study, we investigated HER3 dynamics including HER3 receptor turnover and payload delivery in cancer cells using HER3-DXd both as a single agent and in combination with RTKi including osimertinib, which is in clinical trials in combination with HER3-DXd.

Methods: HER3/ADC internalization was evaluated by using confocal imaging in MDA-MB-453 cells treated with HER3-DXd. Internalization and payload release were quantitatively measured in 3 cancer cell lines treated with HER3-DXd. HER3 turnover on the cell surface was also evaluated upon wash-out of HER3-DXd. In xenograft models, mice were administered two doses of HER3-DXd at different doses and dosing intervals, and membrane HER3 expression and tumor payload concentration were examined over time. NSCLC cell lines harboring EGFR activating mutations, ROS1 fusions, or ALK fusions were used to evaluate the effect of osimertinib, lorlatinib, or ceritinib on cell surface HER3 expression and payload release (osimertinib only).

Results: HER3-DXd was rapidly transferred to early endosomes after binding to HER3. HER3 dynamics varied among the cell lines tested in vitro, and payload release reflected cell surface HER3 expression levels, HER3 internalization speed and turnover rates. In xenograft models, a higher dosage of HER3-DXd resulted in a larger decrease in membrane HER3 expression. Dosing interval also affected membrane HER3 expression levels; the degree of tumor payload concentration increase after the second dose was dependent on the recovery of HER3 expression after the first dose. Furthermore, we confirmed that RTKi increased the cell surface HER3 expression in NSCLC cell lines with targetable driver genomic alterations and that osimertinib increased payload delivery in PC-9 cells through the upregulation of cell surface HER3 expression.

Conclusion: HER3 expression was dynamically changed by HER3-DXd dosing regimen and by RTKi treatment, resulting in a substantial impact on payload release. These findings support our strategy of clinical studies using HER3-DXd after drugs that increase HER3 expression including EGFR TKI and indicate that HER3 dynamics may play a key role in achieving optimal efficacy of HER3-DXd.

Abstract 3997: Preclinical characterization of ARX517, a next-generation anti-PSMA antibody drug conjugate for the treatment of metastatic castration-resistant prostate cancer

Prostate cancer is the most common cancer, and the second leading cause of cancer death, among men in the United States. Metastatic castration-resistant prostate cancer (mCRPC) is an advanced stage of disease in which patients ultimately fail androgen-deprivation therapies and exhibit a poor survival rate. Recently, prostate-specific membrane antigen (PSMA) has been validated as a prostate cancer tumor antigen with its over-expression in prostate tumors and low level of expression in select normal tissues. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX517, an anti-PSMA targeted next-generation antibody drug conjugate (ADC), for treatment of mCRPC patients. ARX517 is composed of a humanized anti-PSMA antibody site-specifically conjugated to drug linker AS269 (a potent tubulin inhibitor), yielding a drug-to-antibody ratio of 2. After binding to PSMA expressed on the surface of tumor cells, ARX517 is internalized and delivers a cytotoxic payload which inhibits tubulin polymerization and induces cellular apoptosis. In vitro testing of ARX517 in prostate cancer cell lines with variable PSMA expression demonstrated highly specific and potent sub-nanomolar activity in cells with high PSMA expression. To minimize premature payload release, ARX517 employs a non-cleavable PEG linker and stable oxime conjugation chemistry to enhance stability in circulation. ARX517 exhibited a long terminal half-life and high serum exposure in mice. The serum stability of ARX517 should effectively deliver more payload to target tumor cells, and in multiple CDX and PDX prostate cancer models, ARX517 showed dose-dependent anti-tumor activity in both enzalutamide-sensitive and enzalutamide-resistant models. Repeat dose toxicokinetic studies in non-human primates demonstrated ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 elicited highly specific, potent cell killing in cell lines with high PSMA expression, inhibited tumor growth in enzalutamide-sensitive and enzalutamide-resistant CDX and PDX models, demonstrated a tolerable safety profile in cynomolgus monkeys, and has a clear therapeutic index based on preclinical serum exposure data. The strong preclinical data and recent clinical validation of PSMA as a mCRPC target provide rationale for evaluation of ARX517 as a potential prostate cancer treatment. ARX517 is currently in a Phase 1 dose escalation trial (ARX517-2011 [NCT04662580]) in the United States.

Abstract 4000: A novel pegylated bispecific antibody-drug conjugate (P-BsADC) targeting Her2+ cancers with improved efficacy and therapeutic window

Despite the fact that ADCs improve the efficacy and target selectivity comparing to the non-specific small molecule cytotoxicity drugs in cancer treatment, traditional ADCs still suffer from many issues which include low tumor penetration and accumulation, inefficient internalization, undesired efflux of ADC from tumor cells, significant on-target off-tumor toxicity, Fc mediated uptake that results in off-target toxicity, limited extravasation across capillary walls due to big molecular size, poor diffusion into the tumor masses due to increased tumor interstitial fluid pressure, and the binding-site-barrier. To address these issues, we previously reported that the compound JY201, a Polyethylene Glycol (PEG)-based bispecific ADC (P-BsADC) targeting two epitopes of Her2, demonstrated advantages in tumor penetration, internalization efficiency, lysosome trafficking effectiveness, no Fc related toxicity, and better efficacy in tumor inhibition than transtuzumab deruxtecan (Ds-8201). Continuing from our previous study, here we further reveal that JY201 can penetrate the tumor deeply and distribute more homogeneously in entire tumor masses while Ds-8201 limits its diffusion to the regions very close to the blood vessels in the tumor. Furthermore, JY201 shows better efficacy than Ds-8201 in inhibiting tumors with low expression of Her2 in pdx (patient derived xenograft) and cdx models. In addition, JY201 can effectively inhibit tumors resistant to Ds-8201. In an in-vitro plasma stability test, JY201 demonstrated high stability in cynomolgus monkey and human serums. JY201 also has a biodistribution profile advocating better safety than Ds-8201 in tumor bearing mice. In the repeated-dosing toxicological study in Her2 transgenic mice, JY201 with the dose of 50mg/kg was well tolerated and did not induce any tissue/organ damage to the animals. Due to much shorter half-life (5 times shorter) in mice for PEGylated proteins than in primates, we expect JY201 will have much higher tolerated dose than the 50mg/kg in primates. In summary, the findings from this study provide solid preclinical evidence for JY201 to be developed further as an efficacious and safe clinical treatment for patients with Her2 positive cancers.

Abstract 4129: Preclinical assessment of GlycoConnect™ ADCs with potency-modulated derivatives of PNU-159,682

PNU-159,682 is an oxidized secondary metabolite of nemorubicin (MMDX), and substantially more potent (2100-6400-fold) than the commonly used chemotherapeutic anthracycline doxorubicin/adriamycin, but without the dose-limiting cardiotoxicity. Based on these beneficial features, PNU-159,682 is currently being clinically evaluated (phase 1) for solid tumor indications as a payload in various antibody-drug conjugates (NBE-002, SO-N102). It must be noted, however, that the HNSTD of current PNU-based ADCs in non-human primates (~1 mg/kg) may end up in a clinical dose that is sub-optimal to achieve high tumor uptake, due to putative target-mediated drug disposition in healthy tissue.

We embarked on a program to develop a set of 2nd-generation PNU-159,682 analogues with attenuated potency, to enable the generation of ADCs with a potentially improved pharmacokinetic profile. Moreover, we reasoned that the adaptation of the new PNU analogues to our proprietary best-in-class ADC technology (GlycoConnect™)1, in combination with our polar spacer technology (HydraSpace™)2 might enable PNU-based ADCs with significantly expanded therapeutic index (TI).

We here exhibit the chemical synthesis of a panel of PNU-analogues with attenuated potency and enhanced tolerability, based on specific tailoring of the aminosugar morpholino group. Homogeneous and stable GlycoConnect™/HydraSpace™ ADCs were subsequently generated and evaluated in vitro and in vivo to assess efficacy and tolerability. These studies demonstrate the potential of attenuated PNU derivatives for application in stable and site-specific ADCs with anticipated higher clinic dose and potentially improved therapeutic index.

Abstract 4151: Anti-CEACAM5 immune stimulant TLR7/8 agonist antibody drug conjugate is a potent myeloid cell activator for the treatment of CEACAM5-expressing tumors

Toll Like Receptor 7 and 8 (TLR7/8) agonists are a promising approach to treat tumors by harnessing the innate immune system to trigger anti-tumor innate and adaptive immunity. One of the main obstacles to the use of this class of low molecular weight molecules in cancer treatment is the toxicity associated with systemic immune activation after intravenous administration. We developed a novel immune stimulant ADC by conjugating the anti-CEACAM5 (carcinoembryonic antigen related cell adhesion molecule 5) tusamitamab antibody with a resiquimod (R848) TLR7/8 agonist payload. This tumor-targeted compound administered systemically and locally active, is expected to eradicate CEACAM5-positive (+) tumors by recruiting and activating immune cells in tumor microenvironment to promote anti-tumor immune response. Tusamitamab R848 ADC, evaluated in vitro, binds to CEACAM5+ tumor cells via its Fab moiety, and to myeloid cells via its Fc part at nanomolar (nM) concentrations. TLR7/8 pathway activation was evaluated on myeloid THP1 reporter cells. The conjugate elicits a potent and FcyR-dependent activity at low nM concentration. In co-culture of human blood cells and human CEACAM5+ gastric cancer cells, it was shown to induce phagocytosis activity, associated with increased activation/maturation of both monocytes and dendritic cells. Tusamitamab R848 ADC evaluated in vivo in CEACAM5+ pancreatic human tumor model, HPAFII, leads to robust antitumor activity with complete regressions (CR) after single administration at 5 mg/kg. In a pharmacodynamic study using lung tumor patient-derived xenograft mouse model, this ADC triggers murine cytokine inductions, innate immune cell enrollment at tumor site, and tumor necrosis in a dose-dependent manner. Based on preclinical in vitro and in vivo data, the tusamitamab R848 ADC is an innovative ADC approach with the potential to eradicate CEACAM5+ tumors in patients.

Abstract 4353: Utilizing scRNA sequencing to understand biomarkers of response and resistance to Sacitizumab Govetican in localized TNBC

Triple-Negative Breast Cancer (TNBC) is an aggressive breast cancer subset, which lacks expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor-2. This subset of breast cancer disproportionately affects Black and African American women and improving TNBC treatment options is vital to reducing breast cancer mortality. The novel antibody-drug conjugate, Sacitizumab Govetican (SG), which targets TROP2 at the cell surface, has shown promising clinical results from the NeoSTAR trial (NCT04230109), a phase II study evaluating neoadjuvant SG therapy in a localized TNBC setting. As part of the NeoSTAR clinical trial, we have collected and processed pre- and post- SG treatment patient samples, with the aim of understanding response to this monotherapy. Herein, we identify biomarkers of response and resistance to SG monotherapy through the use of single cell RNA sequencing of matched pre- and post-treatment patient biopsies combined with exome sequencing these patient samples. Pre-treatment core needle biopsy samples, and if applicable, post-treatment residual disease biopsies were dissociated into single cell suspensions and subjected to single cell RNA sequencing. Additionally, fixed patient tissue samples were processed accordingly for exome sequencing analyses. Overall, we analyzed over 144,000 cells from 37 total scRNA seq libraries with an average of 3800 cells per biopsy sample, demonstrating the feasibility of this method. From these analyses, we observed several cell-type differences between patients who achieved a pathological complete response (pCR) and patients who had residual disease (RD). Specifically, our data shows that tumor infiltrating lymphocytes are a potential prognostic biomarker of response to SG. Furthermore, we detected alterations in among stromal and immune cell subsets, among non-responders, indicating that these cell types maybe indicative of SG resistance. Taken together, we outline biomarkers of response to SG treatment for an improved understanding of resistance mechanisms in the neoadjuvant setting to improve TNBC outcomes among patients.

Abstract 4421: Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity

Introduction: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid lineage cells with potent immunosuppressive activity found enriched in cancer patients. Crosstalk between MDSCs and the tumor microenvironment can promote tumor immune evasion and thus there is growing interest in developing therapeutic approaches to intervene in the MDSC suppressive function. Both monocytic-MDSC (M-MDSC) and granulocytic-MDSC (G-MDSC) subpopulations express the cell surface CD33 myeloid marker. For well-defined cell surface markers like CD33, there is considerable interest in the use of radionuclides as therapeutic payloads, particularly α-particle emitters such as actinium-225 (225Ac) since they deliver substantially higher decay energies over a much shorter distance than β-emitters, rendering them more suitable for precise, potent, and efficient target cell killing while minimizing toxicity to surrounding bystander cells. Actimab A, the anti-CD33 antibody lintuzumab armed with the 225Ac radioisotope (CD33 ARC), is currently being evaluated in R/R AML and has demonstrated significant anti-leukemic activity in Phase 1/2 clinical trials. We therefore hypothesized that MDSCs can be directly targeted by the CD33 ARC. Hence, we evaluated the therapeutic potential of the CD33 ARC to deplete MDSCs through preclinical studies in vitro and in vivo with humanized mouse model.

Methods: CD33 ARC was generated by conjugating lintuzumab with p-SCN-Bn-DOTA and subsequently radiolabeled with 225Ac. Primary MDSCs were isolated from cancer patient peripheral blood or healthy donor PBMCs. The specific binding of CD33 ARC to CD33 positive MDSCs and the decreased viability of MDSCs in response to treatment in vitro was characterized by immunophenotyping using flow cytometry. The therapeutic efficacy of CD33 ARC to deplete MDSC in vivo was evaluated in human CD34 reconstituted humanized NOG-EXL mice.

Results: CD33 positive MDSCs (M-MDSCs and G-MDSCs) were identified in human cancer peripheral blood samples, including colorectal and lung. Significantly more MDSCs were found in the peripheral blood of cancer patients in comparison to healthy donors. CD33 ARC treatment of human cancer MDSCs induced a potent dose-dependent reduction in MDSC viability leading to increased depletion of MDSCs. Furthermore, in the humanized NOG-EXL mouse model, CD33 ARC therapy demonstrated in vivo activity of CD33 ARC to deplete human MDSCs.

Conclusions: In this study, we demonstrate CD33 ARC alpha targeted radiotherapy depletes human CD33 positive immune suppressing MDSCs present in multiple cancer types, to enhance antitumor immunity. These findings present a translatable strategy that supports further evaluation of 225Ac lintuzumab as a MDSC targeting agent to improve the efficacy of antitumor therapies.

Abstract 4423: XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses

XMT-2056 is a systemically administered Immunosynthen STING agonist antibody-drug conjugate (ADC) that targets a novel HER2 epitope and induces complete tumor regressions with a single dose in multiple tumor models. We have previously shown that XMT-2056 delivers its STING agonist payload into tumor cells and FcγRI (CD64)-expressing myeloid cells, activating STING signaling in both cell types, leading to type I interferon (IFN) and anti-tumor innate immune responses. Here, we demonstrate that XMT-2056 exhibits ADCC (antibody-dependent cell-mediated cytotoxicity) function, which synergizes with STING pathway activation and induces potent cancer cell-killing activity in co-cultures of HER2-expressing cancer cells and FcγRIII+ (CD16+) immune cells. We show that both XMT-2056 and HT-19 (the unconjugated parental antibody) retain significant cancer cell-killing activity in an Fc-dependent manner in PBMC co-cultures depleted of FcγRI-expressing myeloid cells. This activity is abrogated by co-depletion of FcγRIII+ immune cells, illustrating the ADCC function of XMT-2056. In this setting, XMT-2056 cancer cell-killing activity was significantly increased compared to HT-19, suggesting that the STING payload contributes to the differential activity observed with XMT-2056 treatment. Indeed, co-treatment of cancer cell and immune cell co-cultures with HT-19 and free STING agonist payload enhanced the anti-tumor responses, although to a lesser extent than XMT-2056, suggesting a synergy between the ADCC function and STING pathway activation. Consistently, XMT-2056 treatment of HER2-expressing cancer cells co-cultured with unstimulated CD56+/FcγRIII+ NK cells induced granzyme b and IFN-γ cytokine production, expression of NK cell activation markers, and cancer cell-killing activity. The ADCC activity of HT-19 was comparable to that of trastuzumab in NK cell co-cultures. Finally, we found that depletion of FcγRI+ cells inhibited the cancer cell-killing activity of XMT-2056 in cancer cell and PBMC co-cultures more substantially compared to depletion of FcγRIII+ cells or CD56+ NK cells, indicating a greater contribution of myeloid cells to the XMT-2056 mechanism of action in this setting. Notably, XMT-2056 was capable of engaging both FcγRI+ myeloid cells and FcγRIII+ NK cells, activating both STING-mediated innate immune responses and ADCC function in triple cultures with HER2-expressing cancer cells. Collectively, our data reveal a synergy between ADCC function and STING pathway induction both mediated by XMT-2056, which enhances the cancer cell-killing activity of FcγRIII+ cells. This additional mechanism of action of XMT-2056 can potentially impact the overall anti-tumor immune responses in tumors infiltrated by FcγRIII+ cells.

Abstract 4436: A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1

It is well-established that T cells play a key role in the success of cancer immunotherapies. The goals of T cell-targeting therapies have been to restore the effector function of tumor-specific T cells that are either dysfunctional due to the immunosuppressive mechanisms in the tumor microenvironment, the lack of co-stimulatory signals, or negative regulation by expression of checkpoint molecules such as PD-1. E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) has recently gained attention as a master regulator of multiple immune-activation mechanisms. Inhibition of CBL-B enables the activation of antigen-specific T cells, and has been shown in syngeneic mouse tumor models to be efficacious as a cancer immunotherapy agent. CBL-B inhibition has also been shown to enhance and prolong the effects of anti-PD-1 antibody (Ab) therapy. However, genetic knockout of CBL-B in mice led to spontaneous autoimmunity characterized by auto-antibody production and infiltration of activated T cells and B cells into multiple organs that could result in tissue damage. In order to mitigate safety issues that could result from systemic CBL-B inhibition, we developed novel antibody-drug conjugates (ADCs) that target CBL-B inhibitors (CBL-Bi) to T cells via binding to PD-1. Using PD-1-expressing Jurkat NFAT reporter cells and mixed lymphocyte reaction assay, we demonstrated that our TPS2 approach enhanced the activation of T cells in vitro compared to treatment with anti-PD-1 Ab alone. Evaluation of pathways downstream of TCR activation in primary exhausted T cells showed increased Notch1, phospho-PLCγ2, and ZAP-70 accumulation following anti-PD-1/CBL-Bi treatment and is correlated with IFNγ induction. When autologous mature dendritic cells were co-cultured with dissociated tumor cells from melanoma patients, the highest magnitude of tumor-infiltrating lymphocyte activation was observed using delivery of CBL-Bi by anti-PD-1 ADC, compared to CBL-Bi alone, anti-PD-1 Ab alone, or combination treatment. The potential of anti-PD-1/CBL-Bi ADC for activation effector T cell function is also evident by increased intratumoral transcript levels of Granzyme B and Perforin following treatment in a humanized mouse model. In conclusion, we validated that the biological activity of CBL-Bi is retained after conjugation with an anti-PD-1 antibody. The use of anti-PD-1 Ab for developing the ADC not only targets the CBL-Bi payload to exhausted T cells but also blocks negative regulation of T cells via PD-1. Together, anti-PD-1/CBL-Bi ADC enhanced T cell activation in vitro as well as in vivo. Evaluation of safety and efficacy in animal tumor models is ongoing, to support further development of the TPS2 approach.

Abstract 4493: Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate for the treatment of sortilin-positive (SORT1+) TNBC and Her2-positive breast cancer

Over the last decade, the targeting of specific cell surface receptors such as human epidermal growth factor receptor 2 (Her2) in breast cancer cells has allowed the emergence of innovative strategies for the delivery of anticancer agents. High expression levels of the sortilin receptor (SORT1) have been reported in various tumors of breast cancer patients, including triple-negative breast cancer (TNBC), HR+ and Her2+ breast cancers. Given SORT1 function in ligand internalization, we addressed the sorting and trafficking of the newly designed TH19P01 peptide recognizing SORT1, and of TH1902, an anticancer TH19P01-docetaxel conjugate (PDC), and whether such PDC could target SORT1+ breast cancers. In vitro, high expression of SORT1 was found in several TNBC and Her2+ breast cancer cell lines as well as in in more than 88% of cases (H-score >100) from commercial breast cancer tissue microarrays. SORT1-mediated cell surface binding and internalization of TH19P01 was first investigated in TNBC-derived MDA-MB-231 cells. The binding and uptake of Alexa488-labeled TH19P01, at 4 and 37°C respectively, were found significantly reduced upon siRNA-mediated SORT1 silencing. This demonstrates that functional SORT1 processes are required for both cell surface recognition and internalization of TH19P01. Moreover, the internalization of TH19P01 and TH1902 in MDA-MB-231 cells were monitored by fluorescence microscopy using a polyclonal antibody generated against TH19P01. Both TH19P01 and TH1902 showed a rapid uptake and co-localized in the perinuclear region with the late endosomal marker Rab-7 and with the lysosomal marker Lamp-1 within 30 minutes indicating that both compounds are internalized through a receptor-mediated endocytosis pathway. In addition, TH19P01 internalization after 1 hour appeared significantly associated within intracellular compartments of MDA-MB-231 cells whereas Herceptin remained mostly localized at the cell surface of Her2+ BT-474 cells. In vivo, weekly administration of intravenous bolus of TH1902 (35 mg/kg) at an equivalent docetaxel MTD dose (15 mg/kg) led to complete tumor regression, while docetaxel only inhibited tumor growth by half in a murine MDA-MB-231 xenograft tumor model. Furthermore, in mice bearing Her2+ HCC-1954 breast tumor xenografts, TH1902 induced complete tumor regression in contrast to docetaxel and Herceptin. Taken together, these preclinical data demonstrate the high anticancer properties of TH1902 against SORT1+ TNBC and Herceptin resistant Her2+ breast cancers. These results demonstrate that TH1902 can be a promising avenue for personalized therapy in the treatment of all SORT1+ breast cancers.

Abstract 4499: The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model

Tumor-infiltrating immune cells are involved in the control of cancer and are closely related to clinical outcomes. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate (PDC) of the sortilin (SORT1)-binding peptide TH19P01 ester-linked to two docetaxel moieties, has been shown to exert superior anti-cancer activities in multiple cancer models including melanoma syngeneic and xenograft murine models. Melanomas express elevated levels of SORT1 and are considered as one of the most immunogenic tumors where immune checkpoint inhibitors are among the standard of care treatment used. Here, we first questioned whether TH1902 anti-cancer effects involved infiltration of immune cells in a murine SORT1-positive syngeneic, non-immunogenic and highly aggressive B16-F10 melanoma model in C57BL/6 mice. Weekly administration of docetaxel reduced by half the growth of B16-F10 tumor allografts, while administration of TH1902 as a single agent and at equivalent docetaxel doses, was well tolerated and resulted in tumor regression after only 2 treatments. Surprisingly, in this immunologically cold tumor, immunohistochemistry analysis of tumors for immune cell infiltration showed a net increase in total leukocytes (CD45+) infiltration within TH1902-treated tumors compared with docetaxel-treated tumors, especially with for tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Furthermore, the increased staining intensities for Perforin and Granzyme B were indicative of the elevated cytotoxic T and natural killer (NK) cells activity in TH1902-treated tumors. This corroborated with increased caspase-3 apoptotic activity. TH1902 and docetaxel doses were next halved and combined with the checkpoint inhibitor anti-PD-L1. In a first study, the TH1902/anti-PD-L1 combination resulted in increased tumor growth inhibition when compared with both agents alone. Interestingly, TH1902 as a single agent showed better tumor growth inhibition than docetaxel or docetaxel/anti-PD-L1 combination. In a second study, the TH1902/anti-PD-L1 combination significantly increased animal survival over either anti-PD-L1 or TH1902 as single agents (21 days increased median survival compared to 2.5 and 12.5 days respectively). We conclude that the superiority of TH1902 anticancer activity over docetaxel involves, in part, the modulation of infiltrating immune cells within the tumor microenvironment. This is the first demonstration that immune cell infiltration patterns play a pivotal role in the TH1902-associated anti-tumoral response. Combination of TH1902 with checkpoint inhibitors (anti-PD-L1) further reveals that this may lead to improved clinical outcomes in future immunotherapy translational approaches.

Abstract 4502: Aptamer conjugated prostate specific membrane antigen (PSMA) targeting nanobees for prostate cancer prevention and therapy

Melittin (MEL), a major peptide component of bee venom (BV), has shown chemopreventive as well as chemotherapeutic effects against many cancers in preclinical model systems. However, its applicability to humans has met with limited success due to several issues including its toxicity, nonspecificity, degradation, inefficient systemic delivery and limited bioavailability. Earlier, the concept of "nanochemoprevention" i.e. the use of nanotechnology to improve the outcome of cancer chemoprevention is introduced. Then, "non-targeted nanobees" i.e. nanoformulated mellitin is developed to exhibit excellent anti-prostate cancer efficacy in vitro and in animal models coining the concept as ‘Nanobee- Chemoprevention’. Here, we are extending this work and developed nanobees targeted with small molecular entities (A10 2′-fluoropyrimidine RNA aptamers (Apt)), able to bind to prostate specific membrane antigen (PSMA), a transmembrane protein that is overexpressed in prostate cancer (PCa) and evaluated their efficacy in preclinical studies. The Apt-Nanobees led to an enhanced anti-proliferative activity in PCa cell lines compared to the free melittin. The behavior of Apt-Nanobees in modulating apoptosis and cell-cycle, was also determined. Then, in vivo experiments, in mouse xenograft model of prostatic tumor using nanobees with a model of targeted nanosystems, were conducted. The obtained data supported our hypothesis of targeted nanobees enhanced the bioavailability and limited unwanted toxicity of melittin, thus leading to a significant potential for probable clinical outcome.

Abstract 4673: Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma

Background Diffuse large B-cell lymphoma (DLBCL) is typically treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Although most patients can be cured with R-CHOP, up to one-third of them relapses with a dismal outcome in most cases. Numerous approaches have been attempted to improve the treatment outcomes with R-CHOP. Polatuzumab vedotin is an antibody-drug conjugate targeting CD79b, which is ubiquitously expressed on over 90% of B-cell NHL malignancies, including DLBCL. To determine the potential of polatuzumab vedotin as a therapeutic agent, we evaluate its potency across a series of DLBCL patient derived xenograft (PDX) models.

Method We established and characterized a series of DLBCL lymphoma PDX models. Eight DLBCL PDX models were selected for polatuzumab vedotin efficacy study, of which three are GCB DLBCL. Gene expression analysis of these lymphoma PDX models was performed using an Illumina NovaSeq 6000 system following Illumina-provided protocols for 2 × 150 paired-end sequencing. The expression status of CD79b on these DLBCL PDX models was also evaluated by IHC.

Conclusion Our data showed that a single dose of polatuzumab vedotin at just 2 mg/kg could reached a comparable effect with R-CHOP and no significant body weight loss was observed. Our result showed that there is no apparent correlation between polatuzumab vedotin responses (TGI) and the CD79b expression (IHC Score). Although polatuzumab vedotin demonstrated encouraging activity in the treatment of DLBCL, some of the PDX models are also resistant to polatuzumab vedotin treatment. To further investigate the molecular parameters of the relative sensitivity of polatuzumab vedotin. Gene expression profiling of those PDX models and their sensitivity to polatuzumab vedotin were conducted, we found that the expression level of BCL-XL was correlated with reduced sensitivity to polatuzumab vedotin.

Abstract 4867: Effect of FGFR3 activity on Nectin-4 expression in urothelial carcinoma

Bladder cancer is a common malignancy in the U.S. and cause of significant morbidity and mortality. Combination platinum-based chemotherapy has long been the mainstay in the treatment of metastatic urothelial carcinoma (mUC); however, over the last decade there have been significant advances in systemic therapy, which includes the FDA approval of the pan-FGFR inhibitor erdafitinib (approved for tumors with FGFR2/3 alterations) and the antibody drug conjugate (ADC) enfortumab vedotin (which targets the cell surface protein Nectin-4). Despite these advances, most patients with mUC will progress and succumb from their disease, highlighting the need for further therapeutic development. In parallel to therapeutic advances, there have been significant advances in the molecular characterization of UC, including identification of luminal and basal subsets. NECTIN4 and FGFR3 alterations are known to be enriched in luminal subtypes and we have found, via query of the TCGA data, that NECTIN4 expression was significantly higher in FGFR3 altered tumors. Given this association we sought to investigate the effect of FGFR3 activity on Nectin-4 expression. Using erdafitinib, in select luminal UC cell lines (RT112, RT4, SW780) with FGFR3 fusion proteins, we found, much to our surprise, that FGFR inhibition increased Nectin-4 expression. Western blots, probing for Nectin-4, following 24H, 48H, and 72H of treatment with erdafitinib (50 nM) showed a significant increase in protein expression in all three cell lines. Importantly, we consistently saw increased expression of what we believe corresponds to the membrane bound form of Nectin-4 and have now confirm such by flow cytometry and immunofluorescence probing for Nectin-4 in the RT112 cell line. We have also seen statistically significant increases in NECTIN4 on RT-PCR following treatment with erdafitinib; however, we believe that Nectin-4 regulation by FGFR inhibition is not merely transcriptional because the induction of Nectin-4 protein appears significantly greater than the fold increase in mRNA. Notably, in two additional UC cell lines (HT1376 - a luminal cell line with high Nectin-4 expression & UMUC3 - a basal cell line with low Nectin-4 expression), with no alterations in FGFR3, we did not see a significant change in Nectin-4 expression following treatment with erdafitinib. Based on our above results, we hypothesize that FGFR3 inhibition, in tumors harboring FGFR3 alterations, may act as a sensitize agent to Nectin-4 ADC targeted therapy; however, our results are preliminary and further investigation, particularly with in vivo models, is warranted.

Abstract 4890: A novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5 has potent anti-tumor activity in colorectal cancer models

Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed on the cell surface of several epithelial tumors. CEACAM5 is expressed in virtually all colorectal cancer, ~90% of which to high levels while normal tissue expression is limited. The high prevalence of CEACAM5 expression in colorectal tumor cells prompted us to develop an investigational anti-CEACAM5 antibody-drug conjugate (ADC) for the potential treatment of CRC patients. We developed a novel ADC, by conjugating an anti-CEACAM5 antibody with a drug linker to a topoisomerase I inhibitor payload. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The topoisomerase I payload was optimized for potency, reduced PGP efflux and enhanced bystander activity. The novel anti-CEACAM5 topoisomerase I inhibitor ADC binds to CEACAM5 at nanomolar (nM) concentrations and kills CEACAM5-positive colon tumor cells with varying levels of CEACAM5 at sub-nM concentrations with no or very low cytotoxicity towards CEACAM5-negative cells. Mechanistically, the potent anti-tumor activity of the ADC is both mediated by direct internalization, processing, and release of the cytotoxic payload within the CEACAM5-expressing tumor cells, and by a bystander effect mediated by diffusion of the payload to the neighboring CEACAM5-negative tumor cells. The novel anti-CEACAM5 topoisomerase I inhibitor ADC is well tolerated in rats after repeated administration of 30 and 50 mg/kg/day, Q1W x 4. In vivo efficacy of this ADC at 1, 3 and 10 mg/kg (single administration) was evaluated in four CRC patient-derived xenografts (PDXs) models. The conjugate elicits potent and specific and dose dependent antitumor activity with complete regression (CR) at 10 mg/kg in the 4 models. This robust anti-tumor activity was further confirmed in a Single Mouse Trial of 16 CRC PDX models, consisting in the use of one animal per PDX model per treatment arm and for which the evaluation of efficacy was based on the criteria RECIST (Response Evaluation Criteria In Solid Tumors) used in clinic. In these criteria, the overall response rate includes complete response (CR) and partial response (PR) and the a disease control rate includes CR, PR and stable disease (SD). The ADC induces a disease control rate of 95% and an overall response rate of 50% following a single dose of 10 mg/kg. The outstanding anti-tumor activity across CRC PDX models and its favorable safety profile in rats support further evaluation of this investigational novel topoisomerase I ADC in CRC patients.

Abstract 4891: Ifinatamab deruxtecan (I-DXd), a novel B7-H3-targeting antibody-drug conjugate, demonstrates efficient payload delivery into tumor through target-dependent internalization

Ifinatamab deruxtecan (I-DXd; DS-7300) is a novel ADC which consists of an anti-B7-H3 antibody linked with a DNA topoisomerase I inhibiting anti-tumor agent, DXd. I-DXd has demonstrated potent anti-tumor activity in B7-H3-positive preclinical cancer models and showed early signs of promising clinical efficacy in patients with heavily pretreated, advanced solid tumors. ADCs are postulated to exert cytotoxicity specifically in tumors that express the target antigen via target-specific uptake of the compound. Accordingly, effective action of an ADC depends on multiple dynamic steps including antigen-specific binding, internalization, trafficking to lysosomes, linker cleavage and payload release, as well as payload potency. The goal of this research was to characterize I-DXd molecular dynamics in preclinical tumor cell models by investigating the relationship among target expression and dynamics, its translocation to lysosomes, lysosomal enzyme-mediated linker cleavage, and payload release after I-DXd treatment. As a result, I-DXd demonstrated target dependent internalization, efficient trafficking to lysosomes, and subsequent payload release in the studied tumor cell lines. Payload release levels generally correlated with target expression in vitro using a panel of cell lines with varying levels of B7-H3 expression. Also, in cell-line derived xenograft models, tumor tissue payload concentration corresponded to I-DXd binding at the cell membrane. These results suggest the importance of cell surface B7-H3 expression and ADC dynamics that lead to payload release in target expressing tumors to exert anti-tumor activity in non-clinical model. In addition to target-dependent uptake in tumor cells, it was observed in viable human lung tumor slices that B7-H3 was also expressed in several non-tumor cells including cancer-associated fibroblasts, endothelial cells, and macrophages in the tumor microenvironment and I-DXd was shown to be internalized in these B7-H3-expressing non-tumor cells. In summary, this pre-clinical study characterizes the relationship between B7-H3 expression in the tumor microenvironment and I-DXd molecular dynamics. Our results support developing I-DXd as a targeted therapy against advanced solid tumors known to overexpress B7-H3.

Abstract 4892: MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads

Monomethyl auristatin E (MMAE), the payload delivered by vedotin ADCs, exerts its cytotoxic effects via microtubule disruption and induction of ER stress, leading to apoptosis and tumor cell death. In addition, MMAE also has the ability to induce immunogenic cell death (ICD) and immune modulation in the tumor microenvironment (TME) (Liu, 2020; Gray, 2020). Other clinical-stage and approved ADCs incorporate payloads that also cause microtubule disruption (DM1, DM4), or that drive DNA damage through topoisomerase I inhibition (exatecan) as the primary mechanism of action (MOA). While ADCs with different linker payloads produce clinical benefit, their long-term impact on survival and how they pair with PD(L)1 agents may differ based on their ability to elicit immune modulation. In this study we have investigated how these payloads compare to MMAE in their ability to drive immune changes in the TME and describe how these changes could support anti-tumor immunity and improve outcomes in the clinic.Building upon an initial in vitro assessment in which we observed superior induction of ICD markers and underlying ER stress with an MMAE ADC, we utilized a B7H4-expressing MDA-MB-468 xenograft model of triple-negative breast cancer to further characterize the ICD properties of ADC payloads. Tumor-bearing mice were treated with a single dose of B7H4-targeted antibody conjugated to either MMAE, DM1, DM4 or exatecan. Tumors were harvested seven days after treatment and processed for immunohistochemistry (IHC) to assess changes in immune cell infiltration and analyzed by RNA-seq to determine alterations in gene expression. Payload-driven anti-tumor activity was observed with all ADC treatments, each leading to a similar reduction in tumor volume compared to vehicle or unconjugated antibody. IHC staining of tumor sections for F4/80, a mouse macrophage marker, revealed that of the microtubule-disrupting agents, only MMAE significantly increased F4/80+ macrophage infiltration in both the tumor nests and stroma. Increased F4/80+ macrophages in tumor nests and stroma was also observed as a result of B7H4-exatecan ADC treatment. RNA-seq analysis revealed a unique profile for the MMAE conjugate, with an increase in human transcripts encoding cytokine and type I interferon response genes. In summary, the anti-tumor activity of the B7H4-MMAE ADC resulted in recruitment of mouse innate immune cells to the xenograft tumors. Importantly, immune response genes related to ICD were increased following B7H4-MMAE treatment compared to DM1, DM4, and exatecan conjugates. Together, these data suggest that treatment with vedotin ADCs results in robust immunomodulatory changes in vivo that are distinct from other clinical ADC payloads.

Abstract 4893: Evaluation of bystander antitumor effect of Dato-DXd in TROP2 (+) and TROP2 (-) lung cell lines admixed tumor xenograft mouse models

Background: 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 inhibitor payload (DXd) via a stable, tumor-selective, tetrapeptide-based cleavable linker (Nakada T. et al. Bioorg Med Chem Lett., 2016). Dato-DXd demonstrated encouraging efficacy in clinical studies in TROP2 expressing tumors, including NSCLC and TNBC (NCT03401385). Clinical efficacy of Dato-DXd in heterogeneous tumors is hypothesized to be derived not only via effective selective delivery of DXd to TROP2 (+) tumor cells but also DXd diffusion from TROP2 (+) tumor cells to nearby TROP2 (-) tumor cells due to high membrane permeability of the DXd payload, an effect called the bystander antitumor effect. In this preclinical study, the bystander antitumor effect was tested by establishing tumor xenograft mouse models containing admixed TROP2 (+) and TROP2 (-) lung cell lines. The antitumor activity of Dato-DXd was tested in these models to demonstrate the bystander antitumor effect of Dato-DXd.

Materials and Methods: EBC-1 cells and NCI-H526 cells or Calu-6 cells were used as models of TROP2 (+) cells and TROP2 (-) cells, respectively. TROP2 (+) cells and TROP2 (-) cells were mixed in various ratios and inoculated subcutaneously in mice to establish heterogeneous xenograft tumor models with various levels of TROP2 expression. Tumor growth inhibition in each model was evaluated by tumor volume after Dato-DXd or control-ADC administration. Immunohistochemistry (IHC) of DXd and TROP2 were performed in Dato-DXd treated tumors to analyze distribution in TROP2 (+) and (-) tumor cells. Tumor cells that responded to Dato-DXd treatment were identified by IHC assay of γH2AX (an indicator of DNA damage).

Results: TROP2 expression analysis showed that various TROP2 positivity tumor models were established by inoculating various ratios of admixed EBC-1 cells with NCI-H526 cells or Calu-6 cells. Dato-DXd did not inhibit growth of tumors consisting of only TROP2 (-) cells, as expected. In contrast, Dato-DXd showed strong tumor growth inhibition effect on all tumors in which TROP2 (+) cells were inoculated in various levels with TROP2 (-) cells. DXd-IHC analysis showed that Dato-DXd distributed to only TROP2 (+) tumor cells in admixed tumors, however, an increase of γH2AX was observed not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells, suggesting TROP2 (-) tumor cells responded to DXd generated and distributed from Dato-DXd bound to TROP2 (+) tumor cells.

Conclusion: Dato-DXd is effective not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells adjacent to TROP2 (+) tumor cells, thereby demonstrating the bystander antitumor effect of Dato-DXd. This property suggests Dato-DXd should be active in the clinical setting even given potential heterogeneity of TROP2 expression.

Abstract 4894: The anti-ROR1 ADC STRO-003 demonstrates immune-modulating properties that may enhance checkpoint blockade

There is growing evidence that tumor-targeted cytotoxins can also enhance anti-tumor immunity by inducing immunogenic cell death (ICD) in tumor cells and promoting recruitment of immune effector cells. We sought to investigate the immune stimulating potential of STRO-003, an antibody drug conjugate (ADC) composed of an anti-Receptor Orphan Receptor Kinase 1 (ROR1) antibody conjugated to a topoisomerase I targeted exatecan warhead via a cleavable linker. ROR1 is an oncofetal transmembrane receptor with restricted expression in normal tissues. Its overexpression in several cancer indications has been described, including in ovarian, non-small cell lung carcinoma (NSCLC), triple negative breast cancer (TNBC), and hematological malignancies, thus making it an ideal ADC target. We have previously demonstrated potent in vivo activity of STRO-003 in a panel of ROR1-expressing lung cancer patient-derived xenograft (PDX) models. Here we show that the exatecan warhead, SC3386, and STRO-003 ADC induced ICD in vitro as evidenced by presentation of cell-surface calreticulin and release of HMGB1. Consistent with this finding, STRO-003-treated cells elicited monocyte activation in a PBMC co-culture assay. To determine if these immunogenic properties could improve therapeutic efficacy, we evaluated STRO-003 in combination with an immune checkpoint inhibitor in a mouse syngeneic model expressing ROR1. We have demonstrated that STRO-003 in combination with checkpoint inhibition significantly enhances efficacy in a syngeneic mouse model and supports durable anti-tumor immunity. Follow-up vaccination studies were performed to further explore the significance of STRO-003-induced ICD and protective immunity in vivo. These results demonstrate that tumor cells pre-treated with STRO-003 or SC3386 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo.

Abstract 4895: Introduction of a platform for preclinical profiling of drug conjugates: a case study with sacituzumab govitecan

Drug-conjugates are an emerging class of anticancer agents combining the cytotoxic activity of highly potent chemotherapeutic agents with the target specificity of an antibody, a small molecule or a peptide ligand directed against a cancer-associated protein. With nearly half a century of development, profound advancements have been made in the development of these therapeutics. The most advanced are antibody-drug conjugates (ADCs) with currently >100 in development and 11 approved by the FDA. To facilitate preclinical evaluation of novel drug conjugates, Reaction Biology and 4HF Biotec have developed a dedicated platform allowing specific in vitro and in silico analyses for this class of therapeutics. The platform is intended to provide information on the potency of the drug conjugate over the corresponding stand-alone cytotoxin, the cancer entities to be treated, the target engagement and the determinant(s) of cancer cell sensitivity. Here, we present our platform to refine antitumor potential of Sacituzumab govitecan (ScG), a recently approved TROP-2 targeted ADC used for the treatment of triple-negative breast cancer. First, we used the ProLiFiler™ (Reaction Biology), a cell proliferation and survival assay with 140 human cancer cell lines, to establish the antitumor profile of ScG, its payload SN-38 and three other cytotoxins (camptothecin, MMAE and mertansine) to validate the results of our assay. The IC50 obtained for the cytotoxins were next validated using the MoAFinder (4HF Biotec), a tool derived from the NIH COMPARE algorithm that ranks drugs based on the similarity of their growth inhibitory profiles. The analysis confirmed that SN-38 and camptothecin antitumor profiles, as established with the ProLiFiler, correlated best with other topoisomerase-1 inhibitors from our database of more than 1,000 compounds. Mertansine and MMAE correlated best with tubulin inhibitors and were more potent than camptothecin and SN-38. Overall, the response to these cytotoxins was stronger in cell lines from hyperproliferative cancers such as hematological malignancies than in those from solid cancers. Next, by using OMICS data (internal and publicly available), qPCR and flow cytometry connected to drug sensitivity profiles, we will present a differential biomarker screen of ScG vs. SN-38 to identify molecular determinants of sensitivity toward the ADC and its cytotoxin. We will address the expression level of TROP2 and its predictivity for tumor cell sensitivity, screen for predictors of SN-38 response and investigate possible off-target effects in cell lines sensitive to ScG but lacking TROP2 expression. Overall, the study demonstrates the potential of our platform to investigate drug conjugates. The comparative profiling of ScG and its payload allows to identify candidate cancer entities for ScG treatment and to improve patient enrollment into clinical trials using biomarkers.

Abstract 4896: NF1 (neurofibromatosis 1) controls microtubule dynamics and dictates sensitivity to maytansinoids

The tumor suppressor NF1 is classically considered a negative RAS regulator, but sparse evidence suggests additional RAS-independent roles. Early studies suggested an interaction with tubulin, which remains poorly characterized to date but may be of particular therapeutic interest as NF1 is somatically mutated across multiple tumor types. We showed that multiple CRISPR-Cas9-engineeerd NF1 KO HER2+ breast cancer cells (BT-474, SK-BR3, HCC1954) become exquisitely sensitive to the Antibody-Drug Conjugate (ADC) Trastuzumab emtansine (T-DM1); we here investigate the underlying mechanism.TDM1 hypersensitivy was specific to the maytansin microtubule-targeting payload, since it was i) replicated by the naked payload but not the naked antibody; ii) absent with other ADCs (T-DxD); iii) not accompanied by increased TDM1 uptake; iv) associated with increased tubulin-maytansin binding in KO cells, as per Cellular Thermal Shift Assay. The mechanism is likely RAS-independent, as KRAS G12V-overexpression did not alter TDM1 sensitivity. RNAseq revealed that KO cells deregulated genes associated with microtubular dynamics and G2-M transition more strongly upon TDM1-treatement. Multiple KO cells investigated by static and live imaging exhibited marked signs of altered mitosis, with longer G2/M, supernumerary centrosomes, chromosome misalignment and frequent aneuploidy, which could also be inferred in multiple public sequencing datasets (TCGA, MSK IMPACT, AACR GENIE). Based on this, we explored NF1 role on microtubule dynamics. We found several lines of evidence for a direct and mitosis-selective interaction between NF1 and tubulin: i) in Immunofluorescence (IF), NF1 was upregulated in mitosis and colocalized with the mitotic spindle; ii) NF1 co-Immunoprecipitated with tubulin in mitosis-enriched but not asynchronous cells; iii) purified NF1 co-eluted with tubulin in size-selection chromatography; iv) in silico modeling with AlphaFold2 predicted an interaction between NF1 central domains and the alpha-beta tubulin dimer. Crucially, KO cells exhibited severe microtubule hypodynamism in cold-induced depolymerization-repolymerization assays, replicated in multiple cell types and accompanied by imbalanced levels of plus/minus-end microtubule-associated proteins. By IF on cocultured WT/KO live cells, KO cells showed significantly higher GTP-tubulin, known to cause microtubular hyperstability, suggesting the intriguing possibility that NF1 may directly regulate tubulin intrinsic GTP-hydrolyzing activity, similar to its role on RAS. In conclusion, we provide extensive mechanistic evidence for a direct and previously underappreciated role of NF1 in microtubular dynamics, which reshapes our understanding of its tumor-suppressive activity and provides a rationale for pharmacological targeting of NF1-mutated tumors.

Abstract 4957: The targeting drug conjugate Tye1001 displayed its potent anti-tumor efficacy in lymphoma

Multiple drug-targeting strategies have been applied to drug development in order to reduce the toxic side effects of the traditional chemotherapy while enhancing its anti-tumor efficacy. Various peptides, proteins and antibodies are usually used to conjugate cytotoxic agents via acting as drug delivery vehicles. In our previous studies, we identified that peptides displayed their efficacious functions to deliver small molecules or oligo DNA to the target sites through ligand-receptor interactions and quick internalization. In our present study, we attempt to synthesize serial drug conjugates via coupling these compounds to peptide or protein vehicles. Tye1001, one of these conjugates, displayed similar potent anti-proliferation activities in lymphoma cells and many other cancer cells compared to the small molecule itself. In particular, this conjugate significantly enhanced its potent anti-tumor efficacy in xenografts. Meanwhile, this conjugate has much less toxic side effects. In our in vivo assays, the similar results were also observed in multiple other tumors such as gastric cancer and leukemia. Our findings provide a more potential druggable opportunity in the clinical applications of cancer patients and the treatment of different types of cancers.

Abstract 5000: MYTX-011: A novel cMET-targeting antibody drug conjugate (ADC) engineered to increase on-target uptake in and efficacy against cMET expressing tumors

MET alterations can act as an oncogenic driver in non-small cell lung cancer (NSCLC) and elevated cMET expression occurs in many cancers. Antibody drug conjugates targeting cMET (cMET-ADCs) have been developed as a strategy to treat cMET+ tumors irrespective of dependency on cMET signaling. cMET- ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients having the highest cMET levels, indicating tumor cMET levels may be limiting for efficacy. We sought to create an ADC with the potential to benefit a broader population of patients including those expressing moderate cMET levels. Here, we describe MYTX-011, an ADC incorporating the clinically validated vcMMAE linker-payload conjugated to a novel, pH-dependent anti-cMET antibody. We hypothesized that engineering the antibody to rapidly lose affinity at acidic endosomal pH would boost ADC uptake and efficacy in cMET+ tumor cells by avoiding non-productive ADC recycling. We conducted mutagenesis of anti-cMET antibodies, screening for variants that selectively lost binding under acidic conditions, and, in parallel, assessed antibody internalization in cell-based assays. The resulting lead humanized IgG1 antibody was conjugated to vcMMAE at engineered cysteine residues (DAR=2) to create MYTX-011, which exhibited rapid dissociation from cMET at pH5.4 but retained high affinity binding at pH7.4 and 6.4. MYTX-011 showed markedly higher (>3 fold) internalization in cMET+ tumor cells and broader, more potent cytotoxicity across a large panel of cMET+ cancer cell lines in vitro compared to a matched ADC based on the unmodified parent antibody lacking pH-dependent binding, or an in-house version of a clinical stage cMET ADC. These findings translated in vivo where MYTX-011 showed superior efficacy (>3 fold based on dose titration) in NSCLC xenograft models with high (EBC-1) or only moderate cMET expression levels (H1373, H1975) compared to the matched parent ADC or an in-house version of a clinical stage cMET ADC. PK and toxicity studies in cynomolgus monkeys revealed that MYTX-011 exhibited favorable PK characteristics, and a toxicity profile similar to previously described MMAE-based ADCs. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ malignancies than other cMET-ADCs.

Abstract 5036: Single dose treatment with a novel Yttrium-90-labeled high affinity anti-B7-H3 antibody selective for the 4Ig-B7-H3 isoform provides long term survivors for established radioresistant colorectal carcinoma

The immune checkpoint antigen B7-H3 (CD276), expressed on cell surfaces of a variety of epithelial solid tumors, is a type 1 transmembrane protein consisting of an extracellular domain of repeating immunoglobulin constant and immunoglobulin variable domains (IgV, IgC, IgV, IgC), known as the 4Ig-B7-H3 isoform. 4Ig-B7-H3 is the dominant isoform in human cancers and is expressed at much lower levels on normal tissues. Humans also express a sheddable 2Ig-B7-H3 isoform containing only one immunoglobulin variable and constant extracellular domain (IgV, IgC) that is found in circulation (soluble 2Ig-B7-H3) and presents a challenge for systemic targeting of solid tumors. To develop an antibody suitable for fulfilling the promise of B7-H3 as a systemic therapeutic target, herein we report a murine antibody (MIL33B) that demonstrates mid-picomolar affinity to human 4Ig-B7-H3 (Kd, 72 pM) with 8-fold selectivity over 2Ig-B7-H3 (Kd, 580 pM) extracellular domains. Furthermore, MIL33B maintains high affinity to porcine 4Ig-B7-H3 (Kd, 102 pM), selectivity over murine 2Ig-B7-H3 (Kd, 41 nM), facilitating pre-clinical testing, and low cross-reactivity to other B7 family members (Kd, >1 μM). MIL33B-AF495 conjugates showed selective binding by live cell fluorescence microscopy to HeLa and HCT116 cells with high endogenous 4Ig-B7-H3 expression and 4T1, B16F10 and CT26 cells induced to express human 4Ig-B7-H3 compared to HeLa 4Ig-B7-H3 KO cells or murine cell lines with endogenous 2Ig-B7-H3 expression. Compared to uptake in respective KO or vector control tumors, quantitative analysis in vivo with 89Zr-DFO-MIL33B and PET imaging of tumor models demonstrated significantly higher normalized tumor-specific uptake in tumors with high endogenous expression of 4Ig-B7-H3 (HeLa) (3.25 ± 0.65 vs. 0.79 ± 0.13 SUV ratio, * p = 0.02) or those induced to express human 4Ig-B7-H3 (B16F10 and CT26) (3.16 ± 0.30 vs. 1.20 ± 0.69 SUV ratio, ** p = 0.0032; and 3.62 ± 1.62 vs. 1.89 ± 0.85 SUV ratio, *** p < 0.0001, respectively). As a first radio-theranostic treatment application, MIL33B labeled with yttrium-90 (90Y-DOTA-MIL33B), a therapeutic beta-emitter, administered as a single dose I.V. (100 mCi, 3700 kBq), induced complete tumor regression and long-term survival of > 50% of mice harboring established syngeneic radioresistant colorectal CT26 tumors expressing human 4Ig-B7-H3 compared to vector control tumors (* p = 0.0376, log-rank test) 90Y-DOTA-MIL33B-responsive mice developed immunologic memory and depletion assays in vivo demonstrated that CD8b+ cells contributed to the therapeutic efficacy of 90Y-DOTA-MIL33B. These results point to the promise of 90Y-DOTA-MIL33B as a selectively-targeted immune priming agent for radioligand therapy of 4Ig-B7-H3-expressing solid tumors.

Abstract 5040: Novel HER3 targeting antibody radioconjugates, 225Ac-HER3 ARC and 177Lu-HER3 ARC, exhibit potent antitumor efficacy in HER3-positive solid tumors

Background: HER3 is a unique member of the EGFR family that collaborates with other EGFR receptors to induce tumorigenesis and drug resistance. Moreover, HER3 expression is linked to poor survival for patients with solid tumors. Despite HER3 being a rational cancer therapeutic target, no HER3-directed therapies have been approved for clinical use. However, new therapeutic strategies such as antibody drug conjugates (ADCs) are being investigated. Targeted radiotherapy, including radiolabeled antibodies (ARCs), is unique mechanistically by inducing cell death independent of biologic pathway inhibition, and can be efficacious with less toxicity relative to other therapeutic modalities. Therefore, we hypothesized that the cytotoxic effects of alpha (225Ac) or beta (177Lu) emitting radionuclides combined with the specificity of anti-HER3 antibody targeting is a compelling therapeutic approach for HER3-expressing tumors. Here we evaluated the antitumor effects of 225Ac or 177Lu armed HER3 ARCs across multiple HER3-expressing cancer models such as ovarian, colorectal, prostate, and renal cancer.

Methods: ARCs were prepared by radiolabeling AT-02, an anti-HER3 antibody, with 225Ac or 177Lu using p-SCN-Bn-DOTA to yield 225Ac or 177Lu-HER3 ARC. The binding activity and tumor cell cytotoxicity of HER3 ARCs were assessed by ELISA using human recombinant HER3, flow cytometry on HER3-expressing cells, and colony forming assays. To evaluate the antitumor growth effects of 225Ac-HER3 and 177Lu-HER3 ARCs in vivo, preclinical human tumor xenograft models were developed. Mice bearing HER3-positive tumors were dosed with 225Ac-HER3 ARC (0.2 or 0.4 µCi), or 177Lu-HER3 ARC (200 or 400 µCi) and tumor growth and body weight was monitored.

Results: The pharmacological binding properties of HER3 antibody radiolabeled with 225Ac or 177Lu were similar to that of unmodified antibody as demonstrated by HER3 binding ELISA and flow cytometry. HER3 ARCs induced cytotoxicity and inhibited colony formation of HER3-positive tumor cell lines. Significant in vivo human tumor xenograft growth inhibition was observed in response to 225Ac or 177Lu HER3 ARCs compared to control groups (unmodified AT02 or IgG ARCs) in the models studied. No significant loss of body weight was observed in mice treated with HER3 ARCs suggesting that all treatments were well tolerated.

Conclusions: In this study, both 225Ac-HER3 ARC and 177Lu-HER3 ARC demonstrated significant antitumor activity against HER3-expressing tumors in a dose-dependent manner. The HER3 targeted radiotherapy approach that we have undertaken could potentially overcome the limitations of current solid tumor therapies in resistance settings and warrants further evaluation in patients with HER3-expressing tumors.

Abstract 5041: TEM-1 targeted alpha therapeutic [Ac-225]-FPI-1848 induces regression in pre-clinical sarcoma xenograft models

Background: Tumor endothelial marker 1 (TEM-1 or CD248) is a cell surface receptor belonging to a family of C-type lectin transmembrane proteins. Identified as a fetal antigen, expression peaks during embryogenesis but is minimally detected in adulthood. In pathological conditions TEM-1 has been shown to be overexpressed in cancer and fibrosis. In sarcomas, expression occurs throughout the tumor environment in malignant cells, stromal cells and perivascular cells. The first line of treatment for sarcomas is surgical removal; however, many sarcomas arise in inoperable sites prompting a need for novel therapies. Herein, we describe the development of a novel TEM-1 targeting radiotherapeutic, [225Ac]-FPI-1848, in mouse xenograft models of sarcoma.

Methods: [225Ac]-FPI-1848 is a radioimmunoconjugate consisting of a humanized anti-TEM-1 monoclonal antibody conjugated to a proprietary DOTA-based chelate (FPI-1397) and radiolabeled with actinium-225 [225Ac]. [177Lu]-FPI-1835 is the lutetium-177 radiolabeled analogue. The biodistribution and therapeutic efficacy of these radioimmunoconjugates were evaluated in human sarcoma xenograft models using nu/nu mice of Balb/c background. For biodistribution studies, approximately 37 MBq/kg of [177Lu]-FPI-1835 was injected intravenously into mice and tumor/organ radioactivity was quantified ex vivo at timepoints between 4 and 196 h. For therapeutic studies, a single intravenous dose of [225Ac]-FPI-1848 was administered over a dose range of 18.5 to 740 kBq/kg. Tumor size and body weight were monitored for 28 days.

Results: Biodistribution studies with [177Lu]-FPI-1835 confirmed robust tumor uptake with minimal normal tissue uptake in three sarcoma models expressing high (SJSA-1), moderate (SK-N-AS) or low (A673) levels of TEM-1. Furthermore, the degree of uptake trended with TEM-1 expression levels across these models (SJSA1> SK-N-AS> A673). In corresponding therapeutic studies, [225Ac]-FPI-1848 demonstrated dose-dependent efficacy consistent with tumor uptake. In the SJSA-1 model, tumor regression was observed at doses of 92.5 kBq/kg or greater. Tumor regression was observed at doses of 185 kBq/kg or greater in the SK-N-AS model. In the low TEM-1 expressing A673 model, significant tumor regression was only observed at the highest radionuclide dose level (740 kBq/kg). Overall, therapy was well tolerated with no significant loss of body weight or clinical signs of poor health.

Conclusions: [177Lu]-FPI-1835 demonstrated target-dependent uptake consistent with the degree of TEM-1 expression in human sarcoma xenograft models. In turn, corresponding efficacy studies with [225Ac]-FPI-1848 demonstrated strong dose-dependent, and target level-dependent efficacy with no apparent toxicity. These results suggest [225Ac]-FPI-1848 could be a promising therapy for TEM-1 expressing sarcomas.

Abstract 5045: NTSR1-targeted alpha therapeutic [Ac-225]-FPI-2059 induces growth inhibition in a preclinical colorectal tumor model

Background: Neurotensin receptor 1 (NTSR1) is overexpressed in multiple cancer indications that include pancreatic, colorectal and prostate cancers, all of which have limited therapeutic treatment options and unmet medical need. Fusion is developing novel targeted alpha therapeutics (TATs) that enable the specific delivery of high energy alpha particles (actinium-225; [225Ac]) to tumor cells while sparing surrounding normal tissues. The alpha radiation released by TATs causes cell damage through the induction of multiple double-stranded DNA breaks leading to tumor cell death. Here, we describe the therapeutic efficacy of an [225Ac]-conjugated, NTSR1 targeting small molecule in a colorectal cancer tumor model.

Materials and Methods: CT26 colorectal cancer cells overexpressing murine NTSR1 (mNTSR1) were generated by lentiviral transduction. Selected cells were evaluated for stable mNTSR1 expression by an in vitro radioligand binding assay and subsequently implanted subcutaneously into Balb/c mice for in vivo evaluations. FPI-2056 (parent compound) was radiolabeled with either lutetium-177 ([177Lu]-FPI-2057) or actinium-225 ([225Ac]-FPI-2059). Biodistribution assessment studies were conducted in mice bearing CT26-mNTSR1 tumors dosed intravenously with [177Lu]-FPI-2057. Therapeutic efficacy studies were conducted by intravenous administration of single doses of 0.185 - 5.55 MBq/kg of [225Ac]-FPI-2059 (0.1-3 µCi) to animals bearing CT26-mNTSR1 tumors, followed by tumor growth monitoring for 50 days. Study endpoints included tumor volume measurements and impact on animal health status.

Results: Evaluation of [177Lu]‐FPI-2057 biodistribution and excretion revealed rapid renal clearance via urine with a clearance from the blood by 24 h. Uptake of [177Lu]‐FPI-2057 was detected in the CT26-mNTSR1 tumors with a maximum concentration of 7.0 %ID/g at 6 h post-injection, dropping to 4.6 and 2.8 %ID/g at 24 and 48 h post-injection, with 20-fold higher uptake in the tumor vs. blood levels at both 24 and 48h time points. Therapeutic administration of a single dose of [225Ac]-FPI-2059 resulted in dose-dependent tumor growth inhibition at doses above 1.85 MBq/kg of [225Ac]-FPI-2059 (1 μCi), which translated into increased survival compared to control animals.

Conclusion: These results demonstrate that targeted delivery of [225Ac]-FPI-2059 to NTSR1 expressing tumors results in significant growth inhibition and enhanced survival, thereby providing promising preclinical evidence to support the clinical development of [225Ac]-FPI-2059.

Abstract 5047: PSMA-targeted actinium-225 conjugate (225Ac-pelgifatamab) potentiates the antitumor efficacy of darolutamide in androgen-dependent and -independent prostate cancer models

Patients with advanced prostate cancer have few treatment options. While androgen receptor (AR) inhibitors are the standard of care for the systemic treatment, patients often develop resistance despite an initial response. Thus, novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is highly expressed in prostate cancer with limited expression in normal tissues. Therefore, PSMA presents an attractive target for radionuclide therapy and PSMA-targeted therapies have already shown efficacy in the clinical setting. Here, we evaluated the efficacy of 225Ac-pelgifatamab, a PSMA-targeted actinium-225 conjugate, in combination with the AR inhibitor darolutamide in androgen-dependent and -independent prostate cancer models. AR inhibitors are known to upregulate PSMA expression, therefore providing a rationale for combining 225Ac-pelgifatamab with darolutamide and potentially resulting in an enhanced treatment effect.

In vitro, darolutamide induced the expression of PSMA in androgen-dependent VCaP and androgen-independent 22Rv1 cells by more than 10-fold and 2-fold, respectively. The efficacy of darolutamide in combination with 225Ac-pelgifatamab was synergistic in these cells with combination indexes of 0.12 and 0.47, respectively. In the androgen-dependent, patient-derived ST1273 model, a single i.v. injection of 225Ac-pelgifatamab at 75 kBq/kg resulted in a treatment/control (T/C) ratio of 0.22 on day 30, while twice daily (BID) oral (p.o.) treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.29. However, the combination of darolutamide and 225Ac-pelgifatamab resulted in enhanced antitumor efficacy with a T/C ratio of 0.008. Furthermore, 5/10 mice remained tumor-free 86 days after the 225Ac-pelgifatamab injection. In the androgen-independent cell line-derived 22Rv1 model, darolutamide (100 mg/kg, p.o., BID) monotherapy was not efficacious with a T/C of 0.84 on day 16 but a single i.v. dose of 225Ac-pelgifatamab at 150 kBq/kg resulted in a T/C ratio of 0.69. Remarkably, the combination of darolutamide and 225Ac-pelgifatamab resulted in further enhanced efficacy with a T/C ratio of 0.34. Flow cytometric analysis of isolated 22Rv1 tumors showed a 50-fold increase in PSMA expression upon darolutamide treatment compared with vehicle. Taken together, these results suggest that upregulation of PSMA expression contributes to the higher efficacy observed when darolutamide is combined with 225Ac-pelgifatamab.

In conclusion, our results provide a proof of concept for investigating the combination of 225Ac-pelgifatamab with darolutamide in the clinical setting.

Abstract 5108: Overcoming roadblocks of immunotherapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is one of the major causes of cancer-related death worldwide. Immunotherapy has helped to increase the survival of NSCLC patients, but the therapeutic response is limited to 23-26% of patients. While many factors impact immunotherapy failure, we have focused on a few, (a) heterogeneous expression of immune checkpoints (ICs) limiting the use of single immune checkpoint inhibitor (ICI) for everyone, (b) additional ICs responsible for blocking innate immune system and lack of antigen presentation to the T cells, and (c) lack of rationally designed combination therapies.Commonly used immunotherapies for NSCLC block PD1-PDL1 interaction, but the majority of the NSCLC patient does not have high PDL1 expression indicating PD1-PDL1 as a subsidiary mode of immune evasion. So, it is necessary to include additional ICIs in order to achieve adequate objective response in all NSCLC patients. In order to investigate the immune checkpoint expression, we screened several NSCLC patient-derived tumor tissues, and only 33% of them showed high expression of PDL1, whereas 64.6% of patients had high CD47 expression. CD47, an innate immune regulator, is overexpressed in lung cancer cells and delivers an “eat-me-not” signal by binding to signal regulatory protein alpha (SIRPα) in macrophage resulting in diminishing phagocytosis and antigen presentation to the T cells.Combining anticancer drugs with immunotherapy agents has become a popular approach to achieve a less toxic and long-acting objective therapeutic response. Here, we introduce antibody conjugated drug loaded nanotherapeutics (ADNs) consisting of a targeted therapy drug, phosphatidylinositol 3-kinase (PI3K) inhibitor PI103, and decorated with two ICIs for CD47 and PDL1. The ADN particles have been designed based on classical liposomal supramolecular nanoparticles followed by surface functionalization by the specific antibody. The anti-CD47-PDL1-ADN has shown increased cellular internalization and delivery of PI103 than IgG-ADN and anti-PDL1-ADN. The anti-CD47-PDL1-ADN showed low hematotoxicity and higher cell-killing potency for the cancer cells. The bispecific anti-CD47-PDL1-ADNs can block PD1-PDL1 and SIRPα-CD47 immune checkpoint interaction, activating both the innate (macrophage) and adaptive (T cells) immune responses. Reduced tumor growth and higher survival probability have been observed in the syngeneic LLC tumor model for anti-CD47-PDL1-ADN than monotherapy and traditional immunotherapy.In summary, we have introduced a lung cancer treatment strategy that can be an effective therapy for NSCLC patients, irrespective of the PDL1 expression level. The strategy combining bispecific immunotherapy and targeted therapy for activating the innate and adaptive immune systems and delivering targeted therapy drugs can emerge as a significant advance in the treatment of NSCLC patients.

Abstract 5387: Discovery of plasma protein biomarkers associated with overall survival in R/R DLBCL patients treated with loncastuximab tesirine

Plasma proteomics is a non-invasive source of potential biomarkers associated with cancer treatment outcomes, including disease progression and overall survival (OS). Loncastuximab tesirine (lonca) is an antibody-drug conjugate, composed of a humanized anti-CD19 antibody conjugated to a pyrrolobenzodiazepine dimer cytotoxin, for the treatment of relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Here, we investigated the association of plasma protein biomarkers with OS of R/R DLBCL patients prior to treatment with lonca (phase 2 trial, NCT3589469, LOTIS-2). Abundances of 888 plasma proteins, including inflammation, cancer and DNA repair associated markers, were measured for 69 patients with R/R DLBCL from plasma samples collected at baseline. Protein markers predictive of OS were selected using a L1 regularized Cox proportional hazards model (CoxPH) incorporating survival time and censoring status. Protein modules associated with OS were discovered by performing principal component analysis (PCA) on scaled protein abundances. Gene Set Enrichment Analysis (GSEA) was performed using Molecular Signatures Database (MSigDB) sets. We initially applied a regularized COX proportional hazards model to our proteomics dataset to identify proteins associated with OS, which revealed 5 protein markers (associated with decreased survival = REG3A, LAP3; associated with increased survival = NPTXR, C1QTNF1, FLT3) which stratified patients into distinct survival groups (p = 2.3e-4). As a complement to this approach and to extend our biological understanding of this cohort, we then applied PCA to the proteomics data to reveal underlying protein modules associated with survival. Our analysis identified principal component 3 (PC3) to have the strongest association to OS (rho= -0.63, FDR = 8.7e-07); with its top loadings consisting of known markers of cancer progression (top 3 proteins: SIGLEC10, IL6, TNFRSF6B). GSEA on PC3 loadings identified 68 significantly enriched gene sets (FDR < 5%). Gene sets reflective of changes in immune activation, increased cytokine activity and interleukin mediated activation (IL6, IL10) were negatively associated with OS, including activation of the following Hallmark pathways: IL6/JAK/STAT3 signaling and inflammatory response pathways.Overall, our results identify proteomics markers associated with DLBCL survival upon treatment with lonca, highlighting the potential of plasma proteins as a source of relevant biomarkers pending additional validation.

Abstract 5459: Association between stomatitis and treatment efficacy with novel TROP2-directed antibody drug conjugate (ADC), datopotamab deruxtecan, in patients with metastatic cancer

Background: Incidence of toxicity with immunotherapy has been correlated with likelihood of treatment response (Hussaini, Cancer Treat Rev 2021; Schuell, Br J Cancer 2005). However, this phenomenon has not yet been demonstrated with antibody-drug conjugates. Datopotamab deruxtecan (Dato-DXd) is a novel ADC composed of an anti-Trop2 monoclonal antibody, a stable tetrapeptide-based cleavable linker, and a topoisomerase-I inhibitor payload (Bardia, Annals of Onc 2021). Early phase studies have demonstrated significant activity in heavily pretreated patients with metastatic non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). Stomatitis was a common treatment-emergent adverse event (TEAE) observed in clinical trials. We conducted a study to evaluate the association between incidence of stomatitis and treatment response with Dato-DXd and evaluate the impact of steroid mouthwash. We hypothesized that presence and grade of mucositis would correlate with observed treatment efficacy.

Methods: We included all patients treated at one academic institution (Massachusetts General Hospital) with Dato-DXd. Stomatitis was assessed by NCI-CTCAE V5.0. Objective Response Rate (ORR) was assessed by RECIST criteria with independent radiology review. Use of mouthwash was evaluated by secondary review and categorized as primary or secondary prophylaxis. A time-to-event analysis was performed to evaluate the time to onset of stomatitis.

Results: 60 patients, including 19 with MBC and 41 with NSCLC, were treated with Dato-DXd between June 2018-September 2022. The median age of patients was 64 and 41/60 patients were female. The median time to onset of mucositis was 14 days (95% CI 12-42 days). There was no significant difference in incidence of stomatitis between the MBC and NSCLC cohorts. Compared to patients who had progressive disease (PD), patients who had a partial response (PR) or stable disease (SD) were 11 times more likely to have stomatitis (OR 11.0, 95% CI 3.61-44.01, p<0.001). Later in the trial (January 2020 onward), 28 patients received primary steroid prophylaxis - excluding these patients, those who were found to have PR/SD were 8.8 times more likely to develop stomatitis compared to patients who had PD (OR 8.8, 95% CI 1.5-73.3, p=0.023).

Conclusion: The development of stomatitis may be associated with higher likelihood of therapeutic efficacy. Given that these findings are a hypothesis-generating post-hoc exploratory analysis from a single institution, this requires validation in larger studies before drawing strong conclusions and impacting clinical decision making. Further research is also needed evaluate the relationship between TEAEs and therapeutic efficacy with other ADCs.

Abstract 5653: LCB97, an antibody drug conjugate (ADC) targeting a novel tumor antigen for the treatment of multiple solid tumors

LCB97 is an antibody-drug conjugate (ADC) directed against a novel tumor antigen, composed of monomethyl auristatin E (MMAE) as payload (DAR 4) and the humanized IgG1 antibody AFF4 generated by Elthera. AFF4 targets a novel antigen preferentially expressed in various solid tumors including pancreatic cancer, ovarian cancer, colon cancer, breast cancer and melanoma. LCB97 generated by using LCB’s ConjuAllTM technology with AFF4 showed potent cytotoxicity in various cancer cell lines including breast cancer (BC), ovarian cancer (OC), pancreatic cancer (PaC), small cell lung cancer (SCLC), colorectal cancer (CRC) and melanoma. LCB97 also showed excellent anticancer efficacy in several cell line-derived xenograft (CDX) models, including BC, CRC, OC and melanoma. Preliminary toxicity assessment in cynomolgus monkeys (an AFF4 cross reactive species) demonstrated that LCB97 was well tolerated within the estimated therapeutic index of ~20 for repeat dosing. In conclusion, LCB97, generated by combining theAFF4 monoclonal antibody of Elthera and the highly innovative ConjuAllTM platform technology of LegoChem Biosciences is a promising next-generation ADC for the treatment of various solid cancers, including BC, CRC, melanoma, OC and PaC.

Abstract 5736: AZD9592: An EGFR-cMET bispecific antibody-drug conjugate (ADC) targeting key oncogenic drivers in non-small-cell lung cancer (NSCLC) and beyond

De novo and acquired drug resistance can limit the long-term efficacy of targeted cancer therapies such as tyrosine kinase inhibitors targeting key oncogenic drivers like EGFR and cMET. Mechanisms of resistance include secondary mutations of EGFR and cMET and other downstream oncogenic pathways such as KRAS and amplification of alternate growth factor receptors. MET amplification or protein overexpression has been established as the most common mechanism of clinical resistance to EGFR inhibitors such as osimertinib. AZD9592 is a first-in-class bispecific ADC designed to target EGFR and cMET, while overcoming pathway-mediated resistance mechanisms that limit other targeted agents. Here we describe the generation, characterization and preclinical evaluation of AZD9592. The ADC was constructed on the backbone of the clinically validated DuetMab monovalent bispecific IgG platform and was engineered with higher affinity for cMET compared to EGFR (>15 fold), with the aim of reducing EGFR-driven toxicity in normal tissues. The antibody is conjugated via a cleavable linker to a proprietary topoisomerase 1 inhibitor (TOP1i) payload (AZ14170132). The internalization and in vitro cytotoxicity (IC50 in the low nM range) of AZD9592 were found to be optimal when both EGFR and cMET were engaged. When EGFR alone was engaged, cytotoxicity was significantly reduced, consistent with the lower affinity for EGFR. Treatment of cells with AZD9592 induced multiple DNA damage response pathway markers (like ATM, ATR, γΗ2ΑX), consistent with the proposed primary mechanism of action (MOA) of direct tumor-cell killing caused by double strand DNA breaks. AZD9592 monotherapy showed activity in vivo in patient-derived xenograft (PDX) models representing multiple EGFR and cMET expressing tumor types, including both EGFR mutant (m) and wild-type NSCLC and head and neck squamous cell carcinoma. Responses (≥30% regression from baseline tumor volume) were observed across a wide range of clinically relevant dose levels, including a 41% response rate in EGFRm NSCLC tumors treated at the lowest tested dose of 2 mg/kg. AZD9592 combined with osimertinib also showed benefit in PDX models derived from patients who progressed on osimertinib alone, as well as models representing primary resistance (EGFR ex20ins). AZD9592 was well tolerated in cynomolgus monkeys over a 6-week period (dosing every 3 weeks). The key safety findings were limited hematological effects, consistent with the MOA of the TOP1i payload. Plasma pharmacokinetics in cynomolgus monkeys showed an acceptable profile at tolerated doses, in line with other EGFR and cMET directed antibodies. These results demonstrate that AZD9592 has a promising efficacy and safety profile in preclinical models representing diverse opportunities in multiple clinical settings.

Abstract 5737: Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate

AZD9592 is a bispecific antibody drug conjugate (ADC) designed to deliver a topoisomerase 1 inhibitor (TOP1i) cytotoxic payload (AZ14170133) to tumor cells. AZD9592 selectively binds to epidermal growth factor receptor (EGFR) and c-MET, two cell surface receptors highly expressed in solid tumors including non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Here we evaluate the pharmacodynamic activity of the TOP1i payload delivery by AZD9592 in an NSCLC-derived xenograft model using immunohistochemistry (IHC) approaches. Treatment-induced DNA double-strand breaks (DSB) and apoptotic cell death were measured using γH2AX, phospho-RAD-50 (pRAD50), and cleaved-caspase-3 (CC3) across increasing exposure to AZD9592. Furthermore, we report in vivo antitumor efficacy of AZD9592 in a panel of NSCLC and HNSCC patient-derived xenograft (PDX) models that were characterized for somatic tumor alterations, including oncogenic driver mutations in EGFR, tumor cell expression of EGFR and c-MET by IHC and deep-learning based image analysis, and targeted proteomics by mass spectrometry. Results demonstrate dose-dependent increases in pRAD50 and γH2AX upon treatment with AZD9592, signifying induction of DNA damage. Increased CC3 and reduced tumor volume (TV) in all treatment groups compared with control groups supports that AZD9592 induces tumor cell death due to formation of DNA DSB. In PDX experiments, tumor growth inhibition (TGI), defined as ≥30% reduction in TV from baseline after a single dose of AZD9592 8 mg/kg, was observed in 73% (16/22) of EGFR mutant NSCLC models. The models evaluated included tumors with or without prior exposure to EGFR tyrosine kinase inhibitors, and harboring diverse mutational profiles and heterogeneous expression levels of EGFR and c-MET. In EGFR wildtype NSCLC and HNSCC PDX, TGI was observed in 60% (12/20) and 44% (4/9) of models, respectively. IHC demonstrated an association of target expression and response to treatment, suggesting a potential predictive feature of response in tumors with elevated antigen expression. Targeted proteomics demonstrated an association between the expression of SLFN11, a known TOP1i sensitivity marker, and treatment response. Collectively, these results support the hypothesized mechanism of action of AZD9592: TOP1i induced tumor cell death due to formation of DNA DSB, and suggest opportunities in the treatment of tumors with a range of molecular features. AZD9592 is currently in a Phase 1 clinical trial in advanced solid malignancies.

Abstract 5946: OBI-999, an anti-Globo H antibody drug conjugate, exhibits synergistic anti-tumor effect in combination with pembrolizumab

Background: Globo H (GH) is highly expressed in a variety of epithelial tumors with a limited expression in normal tissues rendering it a novel therapeutic target. OBI-999 is an antibody drug conjugate (ADC) consisting of a GH-specific monoclonal antibody conjugated with monomethyl auristatin E (MMAE) through a cathepsin B cleavable linker. MMAE is known to induce immunogenic cell death (ICD) which involves in the activation of cytotoxic T lymphocyte-driven adaptive immunity with long-term immunological memory.

Aim: The aim of this study was to evaluate the synergistic effects of OBI-999 + pembrolizumab on tumor growth suppression in several xenograft tumor models.

Methods: OBI-999-induced ICD was examined in vitro by the detection of damaging-associated molecular patterns (DAMPs) such as calreticulin (CRT), HMGB1, and ATP in incubated Globo H expressing cells. The synergistic effects with the combination of OBI-999 and pembrolizumab as well as the ICD-related immunity were assessed in vivo using advanced severe immunodeficient mice that were reconstituted with human peripheral blood mononuclear cells (PBMCs).

Results: Incubation of OBI-999 with high GH expression cancer cell lines (HCC-1428, NCI-N87 and NCI-H526) and mid GH expression cancer cell line (SW-480) induced the release of DAMPs including CRT, HMGB1, and ATP, in a dose- and time-dependent manner, indicating that OBI-999 is capable of inducing ICD in vitro. In the high GH expression human breast cancer cell line HCC-1428 xenograft model, OBI-999+pembrolizumab exhibited significantly stronger inhibition of tumor growth compared to either OBI-999 or pembrolizumab alone. Similar synergistic effects were observed in other xenograft tumor models including gastric (NCI-N87), small cell lung (NCI-H526), and colorectal (SW-480) cancers. Analysis of tumor-infiltrating lymphocytes (TILs) in HCC-1428 humanized mice showed that OBI-999 + pembrolizumab induced populations of activated cytotoxic CD8 T-cells and mature dendritic cells. Pembrolizumab decreased PD-1 expression on CD8 and CD4 cells, and OBI-999 decreased PD-L1 expression on tumor cells, which reversed the exhausted status of immune cells and alleviated immunosuppression in the tumor microenvironment.

Conclusion: We demonstrated significant synergistic effects of OBI-999 and pembrolizumab in several animal models. These synergistic effects may be attributed to the ability of OBI-999 to induce ICD, as demonstrated by the release of DAMPs in vitro and tumor-specific immunity in vivo, suggesting that OBI-999 can create a tumor microenvironment that enhances the function of pembrolizumab. The results suggest that combination therapy with OBI-999 and immune checkpoint inhibitors is warranted in human clinical studies. OBI-999 is currently in Phase 1/2 clinical trials for the treatment of advanced solid tumors with high GH expression (NCT04084366).

Abstract 6135: Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines

Antibody-drug conjugates (ADCs), comprised of cancer cell-specific antibodies linked to cytotoxic drugs, represent an innovative and promising class of anticancer agents. Due to its amplified expression in various tumor types, the epidermal growth factor receptor (EGFR) presents an attractive target for ADC therapy, and three anti-EGFR ADCs are currently studied in clinical trials. As ADCs depend on receptor internalization for intracellular release of drugs in the targeted cancer cells, studying the dynamics of receptor internalization can aid in their development and optimization. We studied the internalization of EGFR induced by its ligand EGF, as a mimic of ADCs, in cell lines with different surface receptor densities.

BxPC-3 (pancreas) and RT-112 (bladder) cancer cells were seeded in 6-well plates and allowed to adhere for 24 hours. Cells were serum-starved overnight, followed by stimulation with 150 ng/mL EGF for 2, 4, 6, 24 and 48 hours. Internalization was studied by quantification of the number of surface-expressed receptors using flow cytometry with Quantibrite PE beads and EGFR-PE antibody. Moreover, incorporation of the ligand-receptor complex into acidic endo-lysosomes was studied using EGF conjugated to the fluorescent pH sensor pHrodo™ Green. Total EGFR and EGFR phosphorylation levels were assessed by western blot analysis. RNA was isolated and converted into cDNA to perform TaqMan qPCR.

Unstimulated BxPC-3 cells showed higher numbers of EGFR per cell compared to unstimulated RT-112 cells. Since high receptor expression is key for some ADCs to induce effective endocytosis, this may indicate BxPC-3 cells as a more attractive model to study EGFR internalization. Stimulation with EGF induced a strong decrease in surface receptor density for both BxPC-3 and RT-112 cells during the first two hours, suggesting receptor internalization. Accordingly, treatment of BxPC-3 cells with pHrodo™ Green EGF demonstrated the incorporation of ligand-receptor complexes into endo-lysosomes. During following hours, the cell surface expression of EGFR remained stable, whereas it partially recovered after 24 to 48 hours. In contrast, the lysosomal incorporation of EGF-EGFR complexes was not decreased at 24 or 48 hours, suggesting that the increase in surface EGFR may be due to de novo protein synthesis rather than recycling. No increase of EGFR mRNA was, however, detected. Finally, increased phosphorylation of EGFR was observed after two to six hours of stimulation, whereas total EGFR levels were highest in unstimulated and 24- or 48-hour-stimulated cells, which is in concordance with the flow cytometry data.

In conclusion, both BxPC-3 and RT-112 cells show dynamic changes in EGFR internalization upon EGF stimulation, despite differences in EGFR expression levels. These cell lines can therefore successfully be used for internalization studies, which may contribute to future ADC development.

Abstract 6150: AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models

Small cell lung cancer (SCLC) is an aggressive malignancy with critical need for new therapies. While currently treated as a single disease, SCLC is heterogenous, comprised of several transcriptional subtypes. Each of these subtypes has distinct drivers and may warrant unique therapeutic targets. Two potential therapeutic targets for SCLC are the pro-survival proteins BCL-2 and BCL-XL. BCL-2 is overexpressed in ASCL1 (A) and POUF3 (P) subtypes of SCLC. We therefore sought to evaluate efficacy of the dual BCL-2/XL inhibitor AZD0466 in SCLC models and to determine whether transcriptional subtype would predict response. AZD0466 is a novel drug-dendrimer conjugate. The active moiety, AZD4320, is a potent dual inhibitor of BCL-2 and BCL-XL. AZD4320 is covalently conjugated to a 5th-generation PEGylated poly-lysine dendrimer through a hydrolytically labile linker to make AZD0466. AZD0466 has been optimized to deliver efficacy while mitigating potential Cmax-driven on-target toxicities of AZD4320. AZD4320 was active (IC50 ≤0.1 µM) in 9/27 SCLC cell lines. AZD4320 in vitro sensitivity was enriched in cell lines that represented A and P subtypes of SCLC compared to NEUROD1 and YAP1 subtypes. We next profiled AZD0466 in a panel of SCLC patient-derived models: 14 patient-derived xenografts and 10 circulating tumor cell-derived xenografts. AZD0466 monotherapy dosed weekly IV was active in 12/24 SCLC xenografts, driving regressions in 8 models. AZD0466 drove efficacy and cleaved caspase-3 induction in a dose-dependent manner. Similar to in vitro, AZD0466 in vivo efficacy was enriched in subtype-A, driving responses in 10/14 ASCL1 models (7 regression, 3 stable disease). AZD0466 response also correlated strongly with BCL-2 mRNA expression (P<0.0001). AZD0466 outperformed the selective BCL-2 inhibitor venetoclax in 6/10 models. Notably, AZD0466 was active in models resistant to platinum/etoposide chemotherapy, the standard-of-care for SCLC. Together, these data suggest BCL-2/XL inhibition has therapeutic potential in SCLC. AZD0466 is in clinical development. The first-in-human study treated 9 patients with advanced solid tumors (NCT04214093) at doses from 50-200mg, all of which were well-tolerated. The BOR was SD observed in 3 patients (100mg) with 1 patient receiving treatment for 5.5 months. AZD0466 is now under evaluation in patients with hematologic malignancies (NCT04865419 and NCT05205161). AZD0466 has been dosed in 33 patients up to 2400mg. No DLTs have been reported to date. Initial clinical activity has been observed through reduction of bone marrow blasts following AZD0466 treatment. AZD0466 exhibits linear PK, consistent across solid tumor and leukemia patients. The doses tested are in line with preclinical studies in SCLC.

Abstract 6294: CXCR5 is a very promising drug target for the development of antibody-drug conjugates to treat patients with lymphoma

Introduction: The chemokine receptor CXCR5 is highly expressed in tumor cells from different lymphoma types and represents a viable drug target for the development of antibody-drug conjugates (ADC) to treat patients with lymphoma.  

Methods: Immunohistochemistry of human tissues of different lymphoma types were stained using antibodies against human CXCR5, CD20, CD70 and CD79b. Slides were examined by a pathologist and scored for expression. CXCR5 expression on tumor cell lines was analyzed by flow cytometry. Cell surface receptor density was analyzed by Quantibrite ™ PE-beads. Evaluation of antibody internalization was performed using the Operetta High Content Imaging System. NOD/SCID mice were transplanted subcutaneously with lymphoma patient-derived (PDX) tumor specimens and treated with 2mg/kg or 10mg/kg VIP924 CXCR5-ADC or isotype control.

Results: CXCR5 reveals very low to no expression in most tissues except for lymph nodes. Expression of CXCR5 and other B-cell targets like CD19, CD20, CD70 and CD79b was analyzed on patient-derived tumor samples. High expression of CXCR5 was found on naïve and previously treated diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and chronic lymphocytic leukemia (CLL) samples. Based on these results, we developed a CXCR5 targeted ADC with a novel, highly potent kinesin spindle protein inhibitor (KSPi) payload linked via a legumain-cleavable linker (VIP924). To compare the performance of KSPi ADCs, the same effector chemistry was attached to antibodies against other B-cell targets and tested in cytotoxicity assays in various cell lines. CXCR5-targeted ADC demonstrates significantly higher activity compared to the other ADCs tested except for the CD79b ADC with equal potency. VIP924 was then tested in vivo in different PDX tumor mouse models with different levels of CXCR5 expression. In the LY2264 and HBL-1 DLBCL models, we obtained a tumor growth inhibition of 68% and 100% respectively compared to isotype control. The median survival time for the isotype control group was 29 days and median survival time for the mice treated with 10mg/kg VIP924 could not be determined as all mice survived until the end of the study (Day 37). No effect on body weight or any adverse effects in the VIP924 treated mice were observed.

Conclusions: CXCR5 is a highly attractive target in hematological malignancies such as DLBCL, MCL, and FL due to high protein expression and almost no expression in healthy tissues. CXCR5-targeting ADC with KSPi payload showed high potency and superiority to other B-cell-targeted ADCs in vitro on a broad range of lymphoma cell lines. VIP924 with a novel legumain-cleavable linker showed activity in in vivo PDX models from lymphoma patients. Due to the high CXCR5 expression found in relapsed DLBCL patients, VIP924 may bring promising new treatment options for previously treated patients with lymphoma.

Abstract 6295: Belzupacap sarotalocan, an investigational virus-like drug conjugate, preferentially binds cancer cell glycosaminoglycans associated with the epithelial to mesenchymal transition

Human papillomaviruses (HPVs) are non-enveloped, double-stranded DNA viruses that utilize modified glycosaminogycans (GAGs) such as heparan sulfate proteoglycans, within the epidermal basement membrane as initial attachment factors prior to cell entry and infection. Similarly modified GAGs are also commonly found on the surface of tumor cells, enabling HPV capsids with tumor-selective binding properties. Belzupacap sarotalocan (bel-sar, formerly AU-011), is a virus-like drug conjugate composed of an HPV virus-like particle (VLP) conjugated to a cytotoxic payload (phthalocyanine dye) which upon light activation, causes rapid immunogenic tumor necrosis of bound tumor cells in vivo and has the potential to induce long-term anti-tumor immunity. Bel-sar has been shown to bind to and kill a wide variety of tumor types in preclinical models and is entering a Phase 3 clinical trial for the treatment of early-stage choroidal melanoma. To investigate the precise GAG modifications as well as the overall genetic signature which mediates the binding specificity of bel-sar, a large, multi-tumor type binding screen of bel-sar on 124 cancer cell lines was performed in vitro, where we identified a strong relationship between drug binding and pathways involved in the epithelial to mesenchymal transition (EMT). Additionally, through dose escalation treatments, HeLa subclones were generated which fail to bind and are resistant to the cytotoxic effects of bel-sar. Gene expression profiling and biochemical characterization of these resistant HeLa cell lines demonstrated a strong reduction in the expression of GAGs and a marked down-regulation of TGF-β signaling, ultimately resulting in the induction of a mesenchymal to epithelial transition (MET). Furthermore, gene-gene correlation studies showed that a variety of GAG biosynthesis genes are associated with TGF-β signaling and EMT progression, and that these genes are important determinates of bel-sar binding. Overall, these data suggest that the binding of bel-sar and HPV VLPs is strongly dependent on the expression and modification of GAGs that occurs during the EMT process. As many cancers, especially metastatic tumors, are thought to undergo at least partial EMT, these data provide mechanistic insights into the tumor-targeting ability of HPV VLP-conjugates such as bel-sar and suggest possible applications for such therapies across a wide variety of other tumor types.

Abstract 6296: Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload

C-type lectin domain family 12 member A (CLEC12A, CLL-1) is a single-pass transmembrane protein of 265 amino acids that is found on monocytes and AML blasts. We have created and characterized antibody-drug conjugates (ADCs) based on a humanized CLEC12A specific antibody to determine whether CLEC12A may be exploited as a therapeutic target for AML. The payload is a proprietary PBD prodrug with a beta-glucuronide trigger, and the linker-payload was conjugated through farnesyltransferase-mediated functionalization. The ADC demonstrated sub-nM cytotoxicity in vitro against several CLEC12A-positive cell lines including HL-60 and PL21. Leu234Ala/Leu235Ala (LALA) mutations were employed to reduce Fc gamma receptor binding and to avoid on-target toxicity. The LALA mutation-bearing ADC displayed a nearly 50-fold decrease in cytotoxicity towards CD34+ hematopoietic stem cells. Its potency was reduced but still sufficient with IC50 values of 61 pM and 15 pM against HL60 and PL21, respectively. Regardless of LALA mutation, the ADCs demonstrated potent antitumor activities with a complete regression at a dose of 0.5 mpk against a subcutaneous HL-60 SCID mouse xenograft model. When the LALA-mutated ADC was tested against a disseminated NSGA mouse model of HL-60-luc, all treated animals survived without clinical symptoms for three weeks after treatment, whereas vehicle-treated animals exhibited morbidity 19 days after treatment or 35 days after tumor implantation. The bone marrow of ADC-treated animals appeared to have nearly fully recovered, whereas that of vehicle-treated animals showed necrosis or tumor growth. In cynomolgus monkeys, the LALA-mutated ADC had a half-life of 82 hours at a dose of 0.2 mpk, and target-mediated drug disposition appeared weak or negligible. Our preclinical studies have shown that CLEC12A targeting ADC can be used as a therapeutics for treating AML.

Abstract 6297: Rationale for the development of a differentiated Trop2 ADC

Background: Trophoblast antigen 2 (Trop2) or Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) is a transmembrane glycoprotein that is overexpressed in several solid tumor cancers. Antibody drug conjugate (ADC) targeting of Trop2 has been clinically validated. The Trop2 ADC Trodelvy® (also known as Sacituzumab govitecan/IMMU-132) is approved for therapy of TNBC and urothelial cancer, and Datopotamab DXd (DS-1062) is currently being tested in NSCLC.

Peak Bio’s Thailanstatin suite of immunomodulatory linker-toxins (L-Ts) is a set of 7 related molecules with distinct ADC features that have been extensively characterized in vitro and in vivo as Her2 ADCs and is being used as a platform to generate a pipeline of potentially differentiated ADCs (AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832).

Results: M2.8 is a fully humanized Peak Bio antibody (Ab) that targets human Trop2 (hTrop2) with high binding affinity [Kd (M)= 8.97-09]. M2.8 was optimized for binding affinity and humanization relative to its parent M2.1 [Kd (M)= 1.78-08], and, both M2.1 and M2.8, display species-specificity for the human and cynomolgus monkey Trop2 proteins. When bound to hTrop2-positive cell lines, M2.1 and M2.8 can rapidly internalize and deliver an ADC payload.

To identify the optimal release for therapeutic efficacy, we conjugated our family of L-Ts with hRS7 at a comparable drug-to-antibody ratio (DAR) of 4. Maximal tumor growth inhibition (TGI) was associated with the Trop2 ADCs that were conjugated via amine coupling to lysine residues using non-cleavable L-T L22, later renamed PH1.

To determine the effect of antibody on TGI efficacy, different doses, and DARs of M2.1 PH1 ADC were compared to PH1 ADCs made from other Trop2 Abs. At low dose levels, M2.1 PH1 ADCs exhibited comparable TGI with hRS7 PH1, TINA PH1, and T6-16 PH1 ADCs, but exhibited significantly greater TGI than hRS7 SN38 (IMMU-132), suggesting that payload potency, rather than epitope, was important in targeting Trop2. The results also suggested that a PH1 DAR of 4 was sufficient to obtain 90% TGI with multiple anti-Trop2 Abs.

M2.1 PH1 ADC was safely tolerated by cynomolgus monkeys at 3 and 6 mg/kg Q3W x 3 doses with mild-to-moderate changes in liver enzymes and platelets that reversed to baseline within 7-10 days of administration of each dose.

The optimized M2.8 PH1 DAR4 ADCs killed Trop2-positive cell lines with single-digit nanomolar potency, while exhibiting low off-target cytotoxicity vs normal human skin fibroblasts and Trop2-negative cell lines in vitro. M2.8 PH1 ADC regressed all 200mm3 sized NCI-N87 tumors and 50% of these tumors exhibited long-term regression for 5 months.

Conclusion: The target validation studies suggest that Trop2 PH1 ADCs were sufficiently differentiated in areas of preclinical efficacy and well tolerated in a toxicologically relevant host.

Abstract 6298: A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors

Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some lymphoma, myeloma and some types of solid tumors including melanoma, non-small cell lung cancers and liver cancers. Previous attempt to target ICAM-1 with a monoclonal antibody bersanlimab (BI505, BioInvent) showed that it was well-tolerated but with limited clinical efficacy. To improve the effectiveness of targeting ICAM-1, we developed a novel ICAM-1 antibody drug conjugate (ADC). The ICAM-1 ADC is comprised of a humanized anti-ICAM-1 hIgG1 antibody conjugated to a novel topoisomerase I inhibitor through a cleavable linker at a drug-to-antibody ratio (DAR) of about 8. The antibody moiety of the ADC targets a unique epitope on ICAM-1 which is far from its ligand binding domain. Potent in vitro cytotoxicity of ICAM-1 ADC, with nanomolar to sub-nanomolar EC50, was observed on a panel of hematologic and solid tumor cell lines. In preclinical studies, one or two doses of ICAM-1 ADC at 5-6 mg/kg were generally sufficient to induce tumor regression in multiple cell line-derived xenograft models, including those of lung and liver cancers. In all models tested so far, the novel topoisomerase I inhibitor conjugated ICAM-1 ADC is at least as efficacious as the same ICAM-1 antibody conjugated to the benchmark topoisomerase I inhibitor linker payload deruxtecan (ICAM-1-DXd). Furthermore, in a repeat dose exploratory non-human primate safety study, the benchmark conjugate ICAM-1-DXd (DAR 8) was well-tolerated at 41 mg/kg, the highest dose tested. In conclusion, ICAM-1 is an attractive target for topoisomerase I inhibitor based ADC and warrants further investigation.

Abstract 6299: ZB131 antibody-drug conjugates induce potent antitumor activity

Antibody-drug conjugates (ADCs) have become increasingly adopted clinically, with 13 drugs FDA-approved and over 100 under clinical development. Despite this momentum and major advances in payload and linker technology, ADCs are limited by off-cancer target-mediated toxicities incurred by currently available targets. In contrast to genomic and proteomic strategies, our unique drug discovery approach accounts for a disease’s native context. This led to the identification of cancer-specific plectin (CSP) as not only overexpressed in malignant tissue compared to healthy, but exclusively present on the surface of cancer cells and absent in normal or pre-malignant tissue. A Phase 0 imaging trial (Biodistribution of Novel Imaging for Resectable Pancreatic Cancer - NCT01962909) evaluating a radiolabeled CSP-targeted peptide has revealed that CSP is bioavailable and abundant, with over a million CSP molecules per cancer cell in pancreatic cancer. Previously, we generated ZB131, a humanized monoclonal antibody that targets CSP. ZB131 demonstrated potent monotherapy efficacy in pancreatic, ovarian, and bile duct preclinical cancer models, indications that have high CSP expression and are being evaluated in a first-in-human Phase 1/2 clinical trial (NCT05074472). Here, we show that CSP is an ideal target for an ADC: it is abundantly and selectively expressed in many indications, bioavailable, and its inhibition is predicted to synergize with FDA-approved payloads. ZB131 binds specifically to CSP, rapidly internalizes, displays linear pharmacokinetics in pre-clinical models, and demonstrates a strong safety profile. We describe the development of two ZB131 ADCs, ZB131-MMAE (monomethyl auristatin E) and ZB131-DXd (Deruxtecan), with drug-to-antibody ratios of 3 – 4 and 7 – 8, respectively. After binding, both ADCs are rapidly and specifically internalized by CSP-positive cells resulting in drug payload release and enhancing cancer cell death compared to ZB131 alone. Moreover, we characterize cytotoxic activity in high and low CSP cell lines to evaluate ADC activity in relation to CSP abundance. In preclinical xenograft models, ZB131-ADC enhanced tumor regression compared to controls at clinically relevant doses. Furthermore, anti-huIgG staining revealed selective target engagement by ZB131-ADC. Taken together, these data show that ZB131-ADCs demonstrate potent antitumor activity and support their evaluation in a Phase 1 clinical trial.

Abstract 6300: Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers

Background: Claudin18.2 (CLDN18.2), a tight junction protein normally expressed only on gastric mucosa, is overexpressed in gastric, pancreatic, esophageal, ovarian, lung and other solid tumors. Unlike in normal tissue, CLDN18.2 is exposed on epithelial surfaces in malignancy. There are no approved targeted therapies for CLDN18.2-expressing cancers. An antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) targeting CLDN18.2 with a monomethyl auristatin E (MMAE) payload site-specifically conjugated via a cleavable linker at a drug-antibody ratio (DAR) of 2, EO-3021/SYSA1801, was developed to target CLDN18.2-expressing cancer cells, minimize toxicities and maximize therapeutic index.

Methods: HEK293-CLDN18.2 cells were used to evaluate CLDN18.2-dependent EO-3021 endocytosis, MMAE payload release, inhibition of proliferation and antibody dependent cellular cytotoxicity (ADCC). Cell cycle distribution and caspase-3/7 activity were measured 24 hours after treating BxPC3-CLDN18.2 pancreatic cells with EO-3021 or the unconjugated antibody, EO-3021 mAb. Inhibition of proliferation by EO-3021 was measured on cell lines ectopically expressing medium to high CLDN18.2 and on cell lines with endogenous low to medium CLDN18.2 expression. Xenograft studies evaluating tumor growth inhibition by EO-3021, EO3021 mAb, and cisplatin or gemcitabine were done in gastric (NUGC4; NUGC4-CLDN18.2), pancreatic (Patu8988S; BxPC3-CLDN18.2), and lung (NCI-H460) cancer models.

Results: EO-3021 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on CLDN18.2 expression. EO-3021 (EC50: 172 ng/ml) and EO-3021 mAb (EC50: 130 ng/ml) demonstrated similar levels of ADCC. EO-3021 but not EO-3021 mAb promoted G2/M cell cycle arrest and apoptosis and exhibited potent activity across cell lines with low, medium, and high CLDN18.2 expression (IC50: 7-456 ng/mL). EO-3021 induced tumor regressions with a single dose across low, medium, and high CLDN18.2-expressing in vivo models derived from pancreatic (2-10 mg/kg), gastric (0.5-10 mg/kg), and lung cancers (4 mg/kg), respectively. In contrast, standard of care (SOC) chemotherapies and EO-3021 mAb did not induce tumor regressions across in vivo models.

Conclusions: EO-3021 demonstrated profound in vivo antitumor activity and outperformed SOC in gastric, pancreatic, and lung cancer models. Results from in vitro and in vivo studies highlight the promising therapeutic potential of EO-3021/SYSA1801 for patients with CLDN18.2-expressing cancers. A Phase 1 study is ongoing to evaluate the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of SYSA1801 in patients with CLDN18.2 positive advanced solid tumors (NCT05009966).

Abstract 6301: Post-translational modifications regulate sensitivity to Polatuzumab Vedotin in DLBCL

Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive malignancy of which one-third of diagnosed patients ultimately relapse and die. New treatment paradigms for relapsed-refractory patients are needed. Most DLBCL tumors are reliant on signaling through their cell surface B-cell receptor (BCR) mediated by ITAMs on CD79A and CD79B to drive anti-apoptotic NF-kB and PI3 kinase signaling. In order to understand how surface BCR is regulated, we performed whole genome CRISPR screens in 9 DLBCL cell lines in which we sorted for high and low surface CD79B or added the CD79B targeted antibody-drug conjugate Polatuzumab-Vedotin (POLA-V). Strikingly, independent of genetic or cell of origin subtype, sgRNAs encoding the entire N-linked glycosylation pathway and specific sugar modification by addition of galactose and sialic acid were found to be enriched in high CD79B fractions and depleted in POLA-V treated samples compared to control. Further testing showed that removing sialic acid by treating with a small molecule inhibitor of sialic acid, genetic perturbation of golgi sugar transporters SLC35A1 and SLC35A2, or the a2,6 sialyltransferase ST6GAL1 resulted in more surface CD79B antibody binding with no significant change in CD79B protein abundance. The specificity of the effect of sialic acid loss on POLA antibody binding was confirmed by removing cell surface sialic acid with exogenous sialidase and observing increased binding of POLA, but not other therapeutic antibodies like Rituximab or CD19 FMC-63. Loss of SLC35A1, SLC35A2, and ST6GAL1 synergized with POLA-V but not the drug conjugate MMAE alone, showing a BCR dependent mechanism of sialic acid loss on POLA-V sensitization. In a further cell of origin subtype analysis of the germinal center B-cell (GCB) DLBCL cell lines, loss of the E3 ligase KLHL6 was found to synergize with POLA-V. Importantly, KLHL6 is heavily mutated across all germinal center derived malignancies, is more highly expressed in germinal center B-cells than other members of the B-cell lineage, and plays an unknown role in the development of autoimmune diseases. Further work will give evidence for the target of KLHL6 mediated ubiquitination and will show how this gene can drive lymphoma and autoimmune disease development in the germinal center.

Abstract 6304: Preclinical development of a next generation antibody drug conjugate (ADC) targeting B7-H3 for treatment of solid tumors

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, is widely expressed in multiple cancers and is associated with poor prognosis. YL201 is a novel B7-H3-targeting antibody-drug conjugate (ADC) and built on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform). This ADC is comprised of an anti-B7-H3 human monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker.

The novel linker-payload and site-specific conjugation of YL201 results in a homogeneous and more hydrophilic ADC. In addition, the binding specificity, affinity and avidity of antibodies show no impact after conjugation. In vitro, upon binding to B7-H3, the antigen-ADC complexes are internalized to lysosomal vesicles releasing the toxic payload, whose cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. In preclinical studies, YL201 is effective in causing tumor regressions in both cell line- and patient-derived xenograft (CDX and PDX) mouse models at doses that are well tolerated. The in vivo pharmacokinetics in monkeys showed that YL201 is deemed as ‘highly stable’ in circulation. Safety evaluations demonstrated acceptable safety profile and no off-target toxicity in monkeys. These non-clinical data suggest the stability of linker in circulation but efficiently release the payload in tumors, and eventually, expanded the therapeutic window.

Taken together, preclinical data suggest that YL201 could be a promising treatment strategy for B7-H3 positive cancer patients.

Abstract 6307: A novel pegylated bispecific antibody-drug conjugate (P-BsADCpb-adc) targeting cancers co-expressing PD-L1 and CD47

ADCs have demonstrated improved efficacy and target selectivity comparing to the non-specific small molecule cytotoxicity drugs in cancer treatment. Yet, in solid tumor therapies, effort for further improvement of efficacy and safety has been hindered by the poor tumor penetration of conventional IgG based ADC, low internalization efficiency, undesired efflux of ADC from tumor cells, narrow therapeutic window due to the on-target/off tumor and Fc induced toxicity, etc. To address those issues, we previously reported that the compound JY201, a Polyethylene Glycol (PEG)-based bispecific ADC (P-BsADC) targeting two epitopes of Her2, demonstrated advantages in tumor penetration, internalization efficiency, lysosome trafficking, no Fc related toxicity, and better efficacy in tumor inhibition than transtuzumab deruxtecan (Ds-8201). In this study, we reveal another novel pegylated P-BsADC (JY207) formed by site-specific conjugation of a bispecific single chain fusion protein targeting PD-L1 and CD47 with PEG-MMAE (a pegylated cytotoxic payload MMAE). As expected, the compound JY207 retains all the advantages that JY201 possesses. Furthermore, JY207 does not bind to human red blood cells, but preferentially binds to CD47/PD-L1 double positive tumor cells, thus reduces the possibility of on-target toxicities. In vitro cytotoxicity studies showed that JY207 has strong potencies in CD47/PD-L1 double positive tumor cells, while showing almost no killing effect to CD47 or PD-L1 single positive tumor cells. In CDX models and a PDX model of transplanted tumor tissues from lung cancer patients, the compound demonstrated excellent tumor inhibition at low doses. In an in vitro plasma stability test, JY207 displays high stability in cynomolgus monkey and human serums. Preliminary repeated-dosing toxicological study has found the maximum tolerated dose of JY207 in CD47/PD-L1 double transgenic mice is 50mg/kg. In vivo pharmacokinetics and toxicological studies of JY207 are being conducted in cynomolgus monkeys and are expected to show desirable results. All those findings in this study warrant JY207 as a promising candidate for the clinical development for patients with CD47/PD-L1 double positive cancers.

Abstract 6308: Preclinical development of ABBV-319: a CD19-targeting glucocorticoid receptor modulator (GRM) agonist antibody-drug conjugate (ADC) for the treatment of B-cell malignancies

Introduction: Glucocorticoids are key components of standard-of-care treatment regimens (e.g., R-CHOP, Hyper-CVAD) for several B-cell malignancies. However, prolonged systemic glucocorticoid treatment results in glucocorticoid-associated adverse events and acquired resistance that limit its therapeutic potential. ABBV-319 is a novel CD19-targeting ADC engineered to reduce glucocorticoid-associated toxicity observed with systemic glucocorticoids while possessing three distinct mechanisms of action (MoA) to increase efficacy: 1) antibody-mediated delivery of GRM to activate glucocorticoid receptor (GR) induced cell death in cancer cells, 2) inhibition of CD19 signaling, and 3) enhanced Fc-mediated cancer cell killing via afucosylation of the antibody backbone.

Results: We identified a GRM agonist that is 15 and 150 times more potent at driving GR transcriptional activation and cell death compared to clinical glucocorticoids dexamethasone and prednisolone, respectively. The conjugation of GRM agonist as the payload on ABBV-319 enables potent GRM-driven anti-cancer activity against malignant B-cell lines in vitro as well as in cell-line and patient derived xenograft (CDX and PDX) models in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic glucocorticoids (e.g., prednisolone) at its maximum tolerated dose in mice. The CD19 monoclonal antibody (mAb) also reduced proliferation of a subset of B-cell malignant cell lines through inhibition of the PI3K/AKT pathway. Moreover, afucosylation of the CD19 mAb in ABBV-319 enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payloads. ABBV-319 bound similarly to both V158 (high-affinity) and F158 (low-affinity) FcγRIIIa allotypes and mediated potent ADCC in co-culture assays with human peripheral blood mononuclear cells (PBMCs). Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ hematopoietic stem cell-engrafted NSG-Tg(Hu-IL15) mice, demonstrating that the three MoA (GR-driven cell death, CD19 signaling inhibition, and ADCC) collectively contribute to anti-tumor activity in vivo. ABBV-319 also displayed on-target depletion of normal human B-cells but did not affect peripheral NK cell counts in mice. Furthermore, CITE-seq profiling revealed that ABBV-319 treatment of human PBMCs activated GRM-induced signature genes restricted to B-cells, demonstrating the specificity of CD19-mediated delivery of GRM.

Abstract 6311: ABBV-400: An ADC delivering a novel topoisomerase 1 inhibitor to c-Met-positive solid tumors

Antibody-drug conjugate (ADC) technology is founded on the premise that antigen-mediated payload delivery selectively targets tumor cells and spares normal tissue. While approval of several ADCs has proven this to be a successful therapeutic approach, there remain opportunities to generate optimized ADCs with tailored selection of antigen matched with payload highly active in the target indication. To this end, ABBV-400 has been developed as an ADC that targets c-Met and topoisomerase-1 (Top1), both of which are overexpressed in solid tumors. The antibody is directed to the cell surface tyrosine kinase receptor, c-Met, a key driver of tumor cell growth and development of resistance to EGFR-targeted and other targeted therapeutics. Supporting this, Teliso-V, a c-Met-targeted ADC with MMAE payload, has been granted Breakthrough Therapy Designation in non-small cell lung cancer (NSCLC). To extend activity of a c-Met-targeted ADC to patients with tumors expressing lower c-Met levels and to indications, such as colorectal cancer (CRC), where MMAE-based ADCs have not been effective, a novel topoisomerase-1 inhibitor linker-drug was developed. Screening over 400 linker-drugs identified a unique, potent Top1 inhibitor with a cleavable valine-alanine linker and stable bromoacetamide attachment designed to minimize systemic payload release. Preclinically ABBV-400 exhibits favorable pharmacokinetics, anti-proliferative activity in vitro, and compelling efficacy in NSCLC, gastroesophageal, and CRC xenograft models. Furthermore, ABBV-400 is well tolerated by cynomolgus monkeys with bone marrow and gastrointestinal tract toxicities common to other Top1 inhibitors. This pairing of a novel linker-drug and an antibody with demonstrated clinical activity provides an exciting opportunity to treat patients with c-Met expression in indications sensitive to Top1 inhibition. ABBV-400 has progressed through dose escalation, and dose expansion is currently ongoing in a Phase 1 FIH clinical study (NCT05029882).

All authors were employees of AbbVie during this research. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication

Abstract 6318: Preclinical characterization of ARX305, a next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers

CD70 has emerged as an attractive tumor target with its over-expression in multiple solid and hematological cancers, yet tightly controlled and limited expression in normal cells. In the last decade, multiple anti-CD70 antibody drug conjugates (ADCs) have been developed, but most, if not all, of these ADC programs have been discontinued. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX305, a CD70-targeted next-generation antibody drug conjugate (ADC) to potentially overcome the challenges associated with earlier ADCs and treat patients with cancers over-expressing CD70. ARX305 is comprised of a proprietary, high-affinity, humanized anti-CD70 antibody stably and site-specifically conjugated to AS269, a potent microtubule inhibitor. After binding to CD70 expressed on target tumor cells, ARX305 internalizes and releases the cytotoxic payload pAF-AS269 which inhibits cellular proliferation. In vitro testing across a panel of solid and liquid tumor cell lines showed ARX305 elicited highly potent and specific cell killing across multiple tumor types with medium to high CD70 expression levels. In multiple in vivo CD70-expressing xenograft or disseminated models, ARX305 induced significant tumor growth inhibition or regression whereas unconjugated antibody exhibited poor activity. ARX305 utilizes stable oxime conjugation chemistry, a non-cleavable PEG linker, and a membrane-impermeable payload to minimize premature payload release in circulation and associated off-target toxicity. These properties of ARX305 result in high serum stability, with rodent pharmacokinetic studies confirming a similar exposure profile of ARX305 compared to unconjugated antibody and a long serum half-life. Repeat dose toxicokinetic studies in cynomolgus monkeys demonstrated ARX305 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, the highly potent anti-tumor activity in multiple tumor types and wide therapeutic index of ARX305 support clinical evaluation of this next generation anti-CD70 ADC. ARX305 is currently in a Phase 1 dose escalation study in China, and the IND in the United States is open.

Abstract 6320: CUSP06/AMT-707, a new CDH6-targeting antibody-drug conjugate, demonstrates potent antitumor activity in preclinical models

Cadherin-6 (CDH6), also known as K-cadherin, is a type II classic cadherin molecule that plays an important role in the embryonic development of the kidney, but has very limited expression in adult tissues. It is overexpressed in several human malignancies, primarily in ovarian, renal, but also (to a lesser degree) gastric, thyroid, cholangiocarcinoma and other tumor types. The characteristics of limited expression in normal tissues, high expression in tumor tissues, and rapid internalization upon antibody binding makes CDH6 a well-suited Antibody-drug Conjugate (ADC) target. We developed a novel CDH6-targeting ADC, CUSP06, consisting of a proprietary humanized antibody selective for CDH6, a protease cleavable linker, and an exatecan payload, with a drug-to-antibody ratio (DAR) of 8. CUSP06 selectively bound to cell surface CDH6 and was efficiently internalized into CDH6 positive ovarian and renal cancer cells. CUSP06 exhibited strong antiproliferative activity against several CDH6 positive cancer cell lines in vitro. Furthermore, compared to a DXd-based ADC, a exatecan-based ADC demonstrated improved bystander effect. Treatment with CUSP06 resulted in tumor regression in several CDH6 positive cell line derived xenograft (CDX) models, including PA-1, OVCAR3, and 786-O. In addition, CUSP06 demonstrated potent antitumor activity with tumor regression observed in two CDH6 low and high expressing patient derived xenograft (PDX) models. The preclinical activity of CUSP06 against CDH6-expressing tumors provide compelling support for the clinical development of CUSP06 in CDH6-expressing human cancers. CUSP06 is currently in IND-enabling studies. CUSP06 showed an expected toxicity profile consistent with it's exatecan payload in the ongoing pilot toxicology studies. We plan to initiate a Phase 1 first-in-human clinical trial in the 2nd half of 2023.

Abstract 6325: A novel EGFR x MUC1 bispecific antibody-drug conjugate, BSA01, targets MUC1 transmembrane cleavage products and improves tumor selectivity

Epidermal growth factor receptor (EGFR) and mucin 1 (MUC1) are tumor-associated antigens that are highly co-expressed in esophageal squamous cell carcinomas, non-small cell lung cancers (NSCLC), and triple negative breast cancers, among others. MUC1, a glycoprotein essential for the formation of the epithelial mucous barrier, is hypoglycosylated and dimerizes with EGFR, a potent oncoprotein, in transformed cells. Dual-targeting of both tumor-associated antigens (TAAs) represents a promising therapeutic strategy to treat common malignancies. We generated a fully human anti-EGFR/MUC1 bispecific antibody-drug conjugate (BSA01) from RenLite® humanized, common light chain mice. BSA01 is conjugated with Monomethyl auristatin E (MMAE). BSA01 targets the extracellular domain of MUC1-C (C-terminal domain of MUC1), which is the membrane-bound MUC1 cleavage product that remains after MUC1 is cleaved, providing more access to the tumor antigen in situ. In addition, we generated affinity and internalization optimized binders targeting EGFR that increases the tumor selectivity, to avoid potential skin toxicity due to ubiquitous basal EGFR expression. Internalization assays demonstrate that the anti-EGFR/MUC1 bispecific antibody used to generate BSA01 was endocytosed in tumor cells co-expressing EGFR/MUC1 more efficiently than mono- or bivalent antibodies targeting EGFR or MUC1 alone. When conjugated with payload, BSA01 also exhibited strong anti-tumor efficacy in vitro against gastric, NSCLC and pancreatic cancer cell lines. Moreover, BSA01 showed similar activity in vivo, effectively inhibiting tumor growth in multiple cell-derived xenograft and patient-derived xenograft models. These results suggest that BSA01 has great clinical potential for treating tumors co-expressing EGFR and MUC1.

Abstract 6415: CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel

Background: Regulatory T cells (Tregs) have a strong immunosuppressive function, inhibit anti-tumor immunity, and promote the occurrence and development of tumors. In addition, a high infiltration of Treg cells is associated with poor survival in various types of cancer. Since CD25 is highly expressed on Treg cells, it is a potential target protein for Treg depletion.

Methods: The binding affinity(Kd) of CD25 aptamer to rhCD25 protein was calculated through bio-layer interferometry(BLI). The cell binding of Cy5-labelled CD25 aptamer was detected by flow cytometry, and the internalization was visualized by confocal microscopy. The cell internalization rate was determined using the MFI value of flow cytometry. Competitive ELISA, western blot, and qPCR were performed to investigate the effect of CD25 aptamer on CD25/IL-2 binding. In vitro cytotoxicity of prototype-ApDC was determined by CCK-8 assay. Moreover, CD25-dependent cytotoxicity of prototype-ApDC was evaluated by Karpas299(Treg-like):HuT78(Teff-like) 1:1 co-culture model using FACS analysis.

Results: CD25 aptamer binds human CD25 protein with 4.64nM affinity. CD25 aptamer showed selective binding and internalization to CD25-positive Karpas299 and had no interaction with CD25-negative Daudi. The internalization half-time of CD25 aptamer in karpas299 was determined to 9.9 min (95% CI 8.2~12.6min). Interestingly, CD25 aptamer was identified as IL-2R antagonist by evaluating competitive ELISA and the level of phosphor-STAT5 and downstream mRNA expression of TGF-beta. The IC50 value of prototype-ApDC was 24nM to Karpas299 while >1uM to Daudi. These results show that prototype-ApDC has selective cytotoxicity against CD25-expressing cells. Furthermore, in a study of an in vitro co-culture model mimicking the Treg-rich intratumoral T cell environment, prototype-ApDC treatment induced CD25-specific depletion resulting in escalating the ratio of HuT89/Karpas299 with increasing exposure time.

Conclusions: Our CD25-targeted aptamer and its drug-conjugated ApDC have demonstrated the selective blocking of IL-2R-STAT5 signaling and induction of CD25-positive cell-specific depletion, respectively. Moreover, in the co-culture system of the Treg-like and Teff-like cells, CD25-ApDC also proved its cytotoxic selectivity. Therefore, one potential strategy in cancer patients is to use CD25-ApDC with excellent tumor permeability as an anticancer agent with a mechanism of Treg cell depletion through specific targeting of CD25 overexpressed in tumor-infiltrating Treg cells.

Abstract 6737: Exposure-response and age subgroup analyses to support body-weight (BW) dosing of brentuximab vedotin (BV) in newly diagnosed high-risk classical Hodgkin lymphoma (cHL) in children and young adults (aged 2-21 years [y]): A randomized children’s oncology group phase 3 trial (AHOD1331)

Introduction: High-risk cHL in pediatric patients (pts) is typically treated with chemotherapy and radiation. BV, an antibody-drug conjugate (ADC) directed to CD30, is approved in six adult indications including cHL and in children with previously untreated high-risk cHL. In AHOD1331, BV was incorporated into the standard chemotherapy backbone treatment regimen of doxorubicin (Adriamycin®), bleomycin, vincristine, etoposide, prednisone & cyclophosphamide (ABVE-PC) for cHL to replace bleomycin and its expectant toxicity. BV-AVEPC treatment resulted in a statistically significant improvement in efficacy compared to ABVE-PC; safety profiles were comparable. We sought to examine the exposure-response (E-R) relationships in this pediatric population.

Methods: Pts in the BV-AVEPC arm received 1.8 mg/kg BV (max: 180 mg) every 3 weeks (Q3W) for a max of 5 cycles. Pharmacokinetics (PK) evaluation focused on pts aged <13 y (n=26), as lower BV exposure was previously reported in pts with lower BW. Age and BW subgroup analyses were conducted on the primary efficacy endpoint, event-free survival (EFS); key secondary efficacy endpoints included early response rate (ERR) and key AEs of interest: peripheral neuropathy (PN) and neutropenia. E-R was evaluated in the PK population.

Results: In all, 593 pts (aged 2 to 21 y) with previously untreated high-risk cHL (Ann Arbor Stage IIB with bulk/IIIB/IVA/IVB) were randomized to ≥1 cycle of ABVE-PC (n=297) or BV-AVEPC (n=296). Compared to adults, BW dosing provided similar BV exposure in pts aged 12 to <18 y (median: 54 kg) but numerically lower ADC exposure (31%) in pts aged 2 to <12 y due to their lower BW (median: 21 kg). However, EFS was comparable between pts aged 2 to <12 y and 12 to <18 y in the BV-AVEPC arm (EFS rate at 36 mo: 96.2% [91.1-100%] vs. 92.0% [88.5-95.7%]). Similarly, in the exploratory E-R no trends were observed for ERR.

Similar PN rates (Grade [Gr] ≥3: 5.8% vs 6.4%) and neutrophil count decrease (Gr ≥3: 53.8% vs 48.6.%) with BV-AVEPC were observed between pts aged <12 y and 12 to <18 y. There were no apparent trends in neutropenia across BW groups but slightly elevated PN in pts with BW ≥70 kg (Gr ≥3: 11.3% vs ≤8.2%). Overall, there was no consistent evidence that PN and neutropenia were exposure or age-driven in this pediatric population.

Conclusions: Consistent efficacy was observed in pediatric pts in the BV-AVEPC arm across age groups despite lower exposure in pts 2 to <12 y, with no evidence of exposure-driven PN and neutropenia. For pediatric pts with previously untreated cHL,1.8 mg/kg Q3W BV + AVEPC ≤5 cycles demonstrated a favorable risk-benefit profile. These data support the 1.8 mg/kg Q3W BV dosage and do not indicate a need for dose adjustment by age or BW subgroups. ZZ and DZ contributed equally.

Abstract CT049: VELOCITY-Lung: A phase 2 study evaluating safety and efficacy of sacituzumab govitecan (SG) + zimberelimab (zim) + etrumadenant (etruma) in patients (pts) with advanced or metastatic non-small cell lung cancer (mNSCLC) progressing on or after platinum (PT)-based chemotherapy and checkpoint inhibitors (CPI)

Background: Single-agent chemotherapy is the standard of care (SOC) in pts with mNSCLC progressing on or after PT-based chemotherapy and CPI but is associated with poor outcomes. SG is a Trop-2-directed antibody-drug conjugate. SG monotherapy demonstrated an objective response rate (ORR) of 17%, with a manageable safety profile in 54 pts with mNSCLC who had multiple prior therapies (Heist RS, et al. J Clin Oncol. 2017), and a phase 3 study is currently ongoing in this population (NCT05089734). Zim (anti-PD-1) and etruma (dual adenosine receptor antagonist) are under clinical investigation for anti-tumor activity. Zim+etruma and SG+CPI have been previously studied, with overall manageable safety profiles. Here we describe the design of substudy-02 of the VELOCITY-Lung phase 2 platform study, which will evaluate novel treatment combinations, including SG+zim+etruma, in pts with advanced or mNSCLC progressing on or after PT-based chemotherapy and CPI.

Methods: VELOCITY-Lung (NCT05633667) is an open-label, multicenter, randomized, phase 2 platform study, interrogating multiple diverse mechanisms of targeting tumor cells. Eligibility criteria include pathologically documented stage IV NSCLC, and progression after PT-based chemotherapy and CPI given either in combination or sequentially, including pts who received maintenance CPI for stage III disease. Pts with actionable genomic alterations must have received ≥1 previous targeted treatment. Pts will be enrolled into the preliminary stage treatment arm, SG+zim+etruma. After new treatment arms are added to this stage, or when the study proceeds to the expansion stage, patients will be randomized. The randomization ratio will be determined by the sponsor depending on the number of experimental arms initiated in the expansion stage, the comparator arm, and any newly added preliminary stage treatment arms. Randomization will be stratified by histology and prior therapy for actionable genomic alterations. Dosing is per recommended phase 2 dose or maximum tolerated dose for study agents and pts will continue to receive treatment until progressive disease, death, unacceptable toxicity, or initiation of a subsequent anticancer therapy. The primary endpoint is ORR assessed by investigator per RECIST v1.1. Secondary endpoints include progression-free survival, duration of response, OS, and safety. During the preliminary stage, efficacy will be compared against historical SOC; during the expansion stage, efficacy will be compared with an active comparator arm within the study. Depending on the number of treatment arms being tested in the expansion stage, this study plans to enroll ~23 to 133 patients globally and is open for recruitment.

Abstract CT052: A phase 1/2 randomized study of imvotamab monotherapy and in combination with loncastuximab tesirine in relapsed/refractory non-Hodgkin lymphomas

Background: Recent advances in the treatment of non-Hodgkin lymphoma (NHL) have transformed the landscape and provided significant benefits to patients. Novel agents that target CD19 and CD20 on B cells, such as bispecific T-cell engagers and chimeric antigen receptor T-cell (CAR-T) therapy, have demonstrated benefit for patients in the relapsed and refractory setting. However, these agents can often be associated with significant toxicity and there is a need for new therapies that can provide clinical benefit with a superior safety profile. Imvotamab is a novel CD20 x CD3 bispecific antibody utilizing an IgM backbone. This allows targeting of up to 10 CD20 binding sites for every CD3 site. In preclinical models, imvotamab demonstrated encouraging antitumor activity and stimulated T-cells in a more physiologic manner than IgG-based antibodies, which may reduce adverse events typically associated with T-cell engagers and CAR-T, such as CRS and neurotoxicity. Loncastuximab tesirine is a CD19-targeting antibody-drug conjugate approved by the US FDA and EMA for relapsed DLBCL after 2 lines of systemic therapy. Loncastuximab tesirine has shown activity in both DLBCL and FL. Combining therapies such as loncastuximab tesirine, which targets CD19 and induce apoptosis of cancer cells, with imvotamab’s ability to eliminate CD20+ tumor cells by engagement with T-cells, may improve treatment outcomes among patients with NHL via synergistic mechanisms of action.

Methods: This study is a Phase 1/2, multicenter, single-arm clinical trial of imvotamab as monotherapy and in combination with loncastuximab tesirine for patients with relapsed/refractory NHL. Phase 1a Dose Escalation is complete with no DLTs or neurotoxicity AEs up to 1000mg dose titration. Phase 1b Combination will evaluate imvotamab and loncastuximab tesirine in patients with R/R 2nd line or later NHL. Phase 2 monotherapy Dose Selection is currently ongoing. The Phase 2 component randomizes patients at two different dose levels (100 mg and 300 mg plateau dose) in two separate indications (R/R DLBCL and R/R FL). Patients receive weekly dosing on Day 1, 8, and 15 of each 21- day cycle. Dosing begins at 15 mg and is increased weekly for 3-4 weeks to reach the plateau dose. Patients then stay at the plateau dose until disease progression or unacceptable toxicity. Patients who achieve a response by Week 12 may switch to a less frequent dosing interval of every 3 weeks. Primary endpoints include frequency and severity of adverse events and objective response rate (ORR) based on Lugano criteria. Correlative biomarker studies will evaluate the relationship of clinical benefit with blood and tissue biomarkers. The study is currently open with patients enrolling in Phase 2 at time of submission. Phase 1b Combination is expected to begin enrollment in the first quarter of 2023. Clinical trial information: NCT04082936.

Abstract CT056: A multicenter, open-label phase 1/2 trial evaluating the safety, tolerability, and efficacy of MORAb-202, a folate-receptor-alpha-targeting antibody-drug conjugate in patients with selected tumor types

Background: MORAb-202 (farletuzumab ecteribulin) is an antibody-drug conjugate (ADC) comprised of the humanized antifolate receptor-alpha (FRα) monoclonal antibody, farletuzumab, and the cytotoxic microtubule inhibitor, eribulin, conjugated by a cathepsin B-cleavable linker. MORAb-202 targets the eribulin payload to tumor cells expressing FRα, where internalization leads to lysosomal cleavage of the ADC and intracellular release of eribulin, causing apoptosis, cell-cycle arrest, and bystander effects in adjacent cells. A previous phase 1 study in Japan of MORAb-202 (NCT03386942) demonstrated antitumor activity across multiple tumor types and identified interstitial lung disease (ILD) as an adverse event of interest (Shimizu CCR 2021). An expansion cohort (doses: 0.9, 1.2 mg/kg) in patients with platinum-resistant ovarian cancer (OC) found meaningful efficacy across FRα-expression levels and ILD/pneumonitis (mainly low grade) was the most common adverse event (Nishio ASCO 2022).

Methods: This multicenter phase 1/2 study (NCT04300556) consists of Dose-Escalation and Dose-Confirmation cohorts. In the Dose-Escalation phase, the primary objectives were to evaluate safety/tolerability and determine the recommended phase 2 dose of MORAb-202 in patients with OC, endometrial cancer (EC), non-small cell lung cancer (NSCLC), or triple-negative breast cancer (TNBC). In the ongoing Dose-Confirmation phase, the primary objectives are (1) to further evaluate safety/tolerability and (2) to evaluate preliminary efficacy (Objective Response Rate) in patients with OC or EC. Based on a population pharmacokinetics model (Hayato ASCO 2022), body-surface-area-based dosing is utilized. The initial cohort has enrolled 7 patients at a MORAb-202 25 mg/m2 IV Q3W dose and is ongoing; further enrollment of patients at 25 mg/m2 and 33 mg/m2 will occur following ILD safety evaluation. Tumor assessments will be conducted by investigators using RECIST v1.1 at screening, every 6 weeks for 24 weeks, then every 12 weeks or as needed. Assessments of computed tomography scans for ILD will be conducted by a central expert review board.

Abstract CT058: TROPION-PanTumor03: Phase 2, multicenter study of datopotamab deruxtecan (Dato-DXd) as monotherapy and in combination with anticancer agents in patients (pts) with advanced/metastatic solid tumors

Background: Pts with metastatic epithelial tumors who progress after initial therapy have a poor prognosis; new therapeutic agents could potentially improve outcomes. Trophoblast cell surface protein 2 (TROP2) is a transmembrane glycoprotein that is over-expressed on the cell surface of many epithelial cancers representing a promising target for surface-directed therapeutic modalities. Dato-DXd is an antibody-drug conjugate consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd monotherapy demonstrated encouraging efficacy and safety in pts with lung and breast cancer (TROPION-PanTumor01 study; NCT03401385).

Methods: The phase 2, multicenter, open-label TROPION-Pantumor03 study (NCT05489211) is exploring safety and efficacy of Dato-DXd as monotherapy and in combination with various anticancer agents (e.g. durvalumab, AZD5305, nivolumab, bevacizumab, chemotherapies) that may be active in the tumor types being evaluated. TROPION-Pantumor03 is a master trial comprising independent substudies to enable simultaneous evaluation of the recommended phase 2 dose (RP2D), safety, and preliminary efficacy of Dato-DXd across several tumor types and treatment combinations. Pts with endometrial, gastric, metastatic castration-resistant prostate (mCRPC), ovarian, and colorectal (CRC) cancer are being enrolled. Eligible pts: age ≥18 years, histologically/cytologically documented advanced or metastatic disease, ≥1 measurable target lesion (RECIST 1.1) not previously irradiated (mCRPC substudy: non-measurable bone metastatic disease permitted), and adequate bone marrow and organ function. An FFPE tumor sample must be available for all pts. Prospective selection for TROP2 expression is required in pts with CRC; in all other cohorts, TROP2 expression will be analyzed retrospectively. In all monotherapy cohorts, pts will receive Dato-DXd 6 mg/kg IV infusion Q3W; in the combination cohorts, Dato-DXd 4 or 6 mg/kg Q3W, as appropriate for each combination. Pts will receive treatment until disease progression, unacceptable toxicity, or withdrawal.

Primary Endpoints: Objective response rate, PSA50 response (mCRPC substudy only), and safety/tolerability.

Secondary Endpoints: Progression-free survival, duration of response, disease control rate, best % change in tumor size, pharmacokinetics, and immunogenicity (anti-drug antibodies). Exploratory endpoints incl. overall survival, pt-reported outcomes, and biomarker analysis (incl. TROP2 and PD-L1 expression, tumor mutational profiling, proteomics and ctDNA profiling). Recruitment is ongoing as of Jan 2023.

Abstract CT066: HERTHENA-Lung02: A randomized Phase 3 study of patritumab deruxtecan vs platinum-based chemotherapy in locally advanced or metastatic EGFR-mutated NSCLC after progression with a third-generation EGFR tyrosine kinase inhibitor

Background: Standard therapies for patients with epidermal growth factor receptor-mutated (EGFRm) non-small cell lung cancer (NSCLC) that has progressed after treatment with third-generation EGFR tyrosine kinase inhibitors (TKIs) offer only limited benefit. Human epidermal growth factor receptor 3 (HER3) is often expressed in primary NSCLC tumors, and HER3 expression is commonly observed in patients with EGFR mutations. HER3-DXd is a novel antibody-drug conjugate composed of a human anti-HER3 monoclonal antibody (patritumab) linked to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. HER3-DXd demonstrated efficacy and safety in a phase 1 study in patients with EGFRm NSCLC that progressed following an EGFR TKI and platinum-based chemotherapy (PBC) (U31402-A-U102; NCT03260491). In previously reported results from dose-escalation/expansion, HER3-DXd 5.6 mg/kg demonstrated a manageable safety profile and promising efficacy (confirmed objective response rate by blinded independent central review [BICR] of 39%, median duration of response of 7.0 months, and median progression-free survival of 8.2 months) in a subset of patients (n=44) with advanced EGFRm NSCLC that progressed after ≥1 line of PBC and a third-generation EGFR TKI.

Trial Design: HERTHENA-Lung02 (NCT05338970) is a global, open-label, randomized, phase 3 trial evaluating the efficacy and safety of HER3-DXd vs PBC in patients (≈560) with metastatic or locally advanced nonsquamous NSCLC with an EGFR-activating mutation (exon 19 deletion or L858R) who have received 1 or 2 lines of EGFR TKI treatment including a third-generation EGFR TKI and had disease progression following treatment with a third-generation EGFR TKI. Patients are randomized 1:1 to receive either HER3-DXd 5.6 mg/kg every 3 weeks or 4 cycles of PBC containing pemetrexed (can be continued as maintenance) with cisplatin or carboplatin. Patients are stratified by prior third-generation EGFR TKI treatment (osimertinib vs other), line of prior third-generation EGFR TKI use (first vs second line), region (Asia vs rest of world), and presence of stable brain metastases (yes vs no). The primary endpoint is progression-free survival by BICR (per RECIST v1.1). The key secondary endpoint is overall survival. Other secondary endpoints include investigator-assessed progression-free survival, objective response rate, duration of response, clinical benefit rate, disease control rate, time to response (all assessed by investigator and BICR per RECIST 1.1), safety, and patient-reported outcomes. Enrollment began May 2022 and is ongoing, with sites in Asia, Australia, Europe, and North America.

Abstract CT067: Pembrolizumab with and without sacituzumab govitecan as first-line treatment for metastatic non-small-cell lung cancer (NSCLC) with PD-L1 TPS ≥50%: phase 3 KEYNOTE-D46/EVOKE-03 study

Background: Sacituzumab govitecan is an antibody-drug conjugate comprising an anti-trophoblast cell-surface antigen 2 (Trop-2) antibody coupled to a potent payload, SN-38, via a proprietary, hydrolysable linker. In heavily pretreated patients with metastatic NSCLC, sacituzumab govitecan showed a durable antitumor response and was well tolerated with a 10-mg/kg dose regimen. Monotherapy with the anti-PD-1 monoclonal antibody pembrolizumab is a standard-of-care therapy for patients with previously untreated advanced/metastatic NSCLC with no sensitizing EGFR or ALK alterations and a PD-L1 tumor proportion score (TPS) ≥50%. KEYNOTE-D46 evaluates whether adding sacituzumab govitecan to pembrolizumab monotherapy can improve outcomes in patients with metastatic NSCLC with PD-L1 TPS ≥50%.

Trial Design: In this phase 3, open-label, active comparator-controlled, randomized study, ~614 eligible adults with previously untreated metastatic NSCLC; no EGFR, ALK, or ROS1 alterations; PD-L1 TPS ≥50%; and measurable disease per RECIST version 1.1 will be randomized 1:1 to receive either pembrolizumab 200 mg Q3W for up to 35 cycles plus sacituzumab govitecan 10 mg/kg on days 1 and 8 of each Q3W cycle (no maximum treatment duration) or pembrolizumab 200 mg Q3W for up to 35 cycles. Randomization is stratified by ECOG PS (0 vs 1), histology (squamous vs nonsquamous), and geographic region (East Asia vs North America/Western Europe/Australia vs Rest of World). Treatment continues until PD, death, unacceptable toxicity, or another treatment discontinuation criterion is met, or (for pembrolizumab) completion of 35 cycles. Dual primary endpoints are PFS per RECIST version 1.1 by blinded independent central review (BICR) and OS. Secondary endpoints include ORR and duration of response per RECIST version 1.1 by BICR, safety, and patient-reported outcomes. Radiographic imaging occurs at baseline; weeks 6, 12, 18, and 24 from randomization; every 9 weeks thereafter through week 51; and then once every 12 weeks until BICR-confirmed PD or the start of new anticancer treatment, pregnancy, withdrawal of consent, or death. PD-L1 expression status is assessed at a central laboratory using PD-L1 IHC 22C3 pharmDx (Agilent, Santa Clara, CA). Health-related quality of life is assessed using validated patient-reported outcome instruments including the EORTC Quality of Life Questionnaire-Core 30 and Quality of Life Questionnaire-Lung Cancer 13. AEs are assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. Enrollment started on December 9, 2022 and is currently ongoing.

Abstract CT121: ARX517, an anti-PSMA ADC targeting mCRPC resistant or refractory to standard therapies: A phase 1 dose escalation and dose expansion study (ARX517-2011, NCT04662580)

Background: Targeted therapies against prostate-specific membrane antigen (PSMA) have exhibited promising antitumor activity in prostate cancer. With the FDA’s approval of (177) Lu-PSMA-617, PSMA has been validated as a target to treat metastatic castration-resistant prostate cancer (mCRPC). ARX517 is an antibody-drug conjugates (ADC) composed of a fully humanized anti-PSMA mAb site-specifically conjugated to AS269, a potent tubulin inhibitor, yielding a drug-to-antibody (DAR) ratio of 2. After binding to PSMA on the surface of cancer cells, ARX517 is internalized and delivers a cytotoxic payload which inhibits tubulin polymerization and induces cellular apoptosis. ARX517’s site-specific linkage, stable conjugation chemistry, and non-cleavable linker exhibit a homogenous drug-antibody-ratio, mAb-like biophysical properties, and exceptional stability. Therefore, ARX517 promotes highly specific tumor cell apoptosis with minimal off-target bystander activity.

Trial Design: The Phase 1 study’s dose escalation and dose expansion evaluates the safety, pharmacokinetics, and preliminary evidence of anti-tumor activity of ARX517 in adults with mCRPC. Patients must have mCRPC which is resistant or refractory to standard therapies, including androgen receptor signaling inhibitors, and show disease progression by Prostate Cancer Working Group 3 (PCWG3) criteria. All patients must have adequate organ function and brain metastases must be radiographically stable. Ascending dose levels of ARX517 administered as a single agent will use an i3+3 design in the dose escalation phase. During the dose expansion phase, ARX517 will be administered at the recommended phase 2 dose (RP2D) or maximum tolerated dose (MTD). Exploratory pharmacodynamic assessment includes PSMA-PET/CT imaging. The total number of subjects is dependent on the number of ascending doses and cohort size before dose expansion.

Status: ARX517-2011 began enrolling patients in July 2021. With no dose limiting toxicities through dose level 6 (2.0 mg/kg Q3W), dose escalation remains ongoing. As of January 2023, the APEX-01 study is currently enrolling in cohort 7 (2.4 mg/kg) of dose-escalation. Dose expansion will start after RP2D or MTD.

Abstract CT164: Tisotumab vedotin (TV) in squamous cell carcinoma of head and neck (SCCHN): interim analysis from innovaTV 207

Introduction: TV, a tissue factor (TF)-directed antibody-drug conjugate, is approved under accelerated approval in the US at a dose of 2.0 mg/kg IV administered every 3 weeks (Q3W) for adult patients (pts) with recurrent or metastatic (r/m) cervical cancer who have progressed on or after chemotherapy. TV is also being evaluated in several advanced solid tumors known to express TF, including SCCHN; antitumor activity has been observed at the approved dose level. Previous reports show that modifying the dosing schedule to optimize key PK parameters such as AUC, Cmax, and Ctrough can lead to further improvement in clinical efficacy. A population PK model based on 399 pts across clinical trials suggests that when TV is administered at 1.7 mg/kg IV on Days 1 and 15 of a 28-day cycle (Q2W), pts are predicted to achieve 24% higher AUC, a higher Ctrough level, and a lower Cmax, compared with the approved cervical cancer regimen (2.0 mg/kg Q3W). Here, we report the first analysis of TV 1.7mg/kg Q2W for r/m SCCHN that has progressed after prior platinum combination with or without immunotherapy.

Methods: innovaTV 207 (NCT03485209) is an open label phase 2 multi-center study evaluating TV monotherapy or in combination for advanced tumors, including pts with r/m SCCHN. In Part C of the study, eligible pts could have received up to 3 lines of systemic therapy for r/m disease and must have received prior therapy with a platinum-based regimen and a checkpoint inhibitor (CPI), if eligible. TV was administered at 1.7 mg/kg IV Q2W. The primary endpoint was objective response rate (ORR), and secondary endpoints included safety and tolerability.

Results: At data cutoff (28Nov2022), 15 pts with SCCHN were treated. The median number of prior lines for r/m disease was 2. All pts received prior platinum therapy and majority (93%) received a CPI. 67% of pts received cetuximab and 53% received taxanes, for r/m SCCHN. Confirmed ORR was 40% (95% CI: 16.3, 67.7), with 1 complete response and 5 partial responses. The safety profile was generally consistent with that observed across TV monotherapy clinical studies. 13 pts experienced a treatment-related adverse event (TRAE), most commonly asthenia (n=7), peripheral sensory neuropathy (PSN) (n=7), and vomiting (n=5). 4 pts experienced Grade ≥3 TRAEs. 2 pts discontinued treatment due to an AE (PSN and dry eye, n=1 each). 11 pts received 3 or more cycles of treatment (1 cycle = 28 days).

Conclusions: For pts with r/m SCCHN who have progressed after prior platinum combination and immunotherapy, TV may be a promising treatment option. Preliminary data suggest encouraging antitumor activity at a higher exposure with Q2W administration while maintaining an acceptable safety profile. The innovaTV 207 study is still enrolling and different dosing regimens are currently being evaluated across advanced solid tumors.

Abstract CT175: Safety, tolerability, pharmacokinetics, and antitumor activity of SHR-A1811 in HER2-expressing/mutated advanced solid tumors: A global phase 1, multi-center, first-in-human study

Background: SHR-A1811 is an ADC comprised of a humanized anti-HER2 monoclonal antibody (trastuzumab), a cleavable linker, and a DNA topoisomerase I inhibitor payload. Here we assessed SHR-A1811 in HER2-expressing/mutated unresectable, advanced, or metastatic solid tumors.

Methods: Pts were eligible if they had HER2 positive breast cancer (BC), HER2 positive gastric/GEJ carcinoma, HER2 low-expressing BC, HER2-expressing/mutated NSCLC, or other HER2-expressing/mutated solid tumors, and were refractory or intolerant to standard therapy. SHR-A1811 at doses of 1.0-8.0 mg/kg was given Q3W (IV). The primary endpoints were DLT, safety, and the RP2D.

Results: From Sep 7, 2020 to Sep 28, 2022, 250 pts who had undergone a median of 3 prior treatment lines in the metastatic setting received at least one dose of SHR-A1811 in dose escalation, PK expansion, and indication expansion part. As of data cutoff on Sep 28, 2022, 1 pt experienced DLT. Treatment-related adverse events (TRAEs) were reported in 243 (97.2%) pts. Grade ≥3 TRAEs, serious TRAEs, and treatment-related deaths were reported in 131 (52.4%), 31 (12.4%), and 3 (1.2%) pts, respectively. Interstitial lung disease (AESI) was reported in 8 (3.2%) pts. Exposures of SHR-A1811, total antibody, and the payload were generally proportional to dose from 3.2 to 8.0 mg/kg. ORR was 61.6% (154/250, 95% CI 55.3-67.7) in all pts. Objective responses were observed in pts with HER2 positive BC (88/108, ORR 81.5%, 95% CI 72.9-88.3), HER2-low BC (43/77, ORR 55.8%, 95% CI 44.1-67.2), urothelial carcinoma (7/11), colorectal cancer (3/10), gastric/GEJ carcinoma (5/9), biliary tract cancer (5/8), NSCLC (1/3), endometrial cancer (1/2), and H&N cancer (1/1). Subgroup analyses of ORR are shown in Table 1. The 6-month PFS rate was 73.9% in all pts.

Conclusions: SHR-A1811 was well-tolerated and showed promising antitumor activity in heavily pretreated advanced solid tumors.

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Abstract CT178: GQ1001: A next generation HER2-targeting ADC that exhibits promising early clinical efficacy with excellent tolerance in a multi-center, Phase Ia study

Background: GQ1001 is a novel HER2-targeted antibody-drug conjugate (ADC) that was developed using innovative conjugation technologies coined intelligent Ligase-Dependent Conjugation (iLDC), that can significantly improves homogeneity and biostability of ADC. In preclinical studies, GQ1001 showed a robust anti-tumor response in multiple HER2+ models alone or in combination with HER2 TKIs and chemotherapeutics, and excellent pharmacokinetics and safety profiles in rats and monkeys due to low level of payload release. Herein we report the initial results of the ongoing phase Ia study, which aims to investigate the safety, tolerability, pharmacokinetics and antitumor activity of GQ1001 in subjects with HER2+ advanced solid tumors.

Methods: In phase Ia dose escalation, a modified 3+3 model was adopted to assess the safety, dose-limiting toxicity (DLT) and maximal tolerable dose (MTD) or dose recommended for dose expansion (DRDE) of GQ1001. GQ1001 was administered intravenously as a monotherapy on Day 1 of 21-day cycles. The starting dose was 1.2 mg/kg, followed by 2.4, 3.6, 4.8, 6.0, 7.2 and 8.4 mg/kg.

Results: As of Dec. 28th, 2022, 32 subjects with HER2-positive advanced solid tumors, predominantly in breast (9), gastric or gastro-esophageal junction (9) and salivary gland (4), were enrolled and received GQ1001 treatment. Patients had a median 3 (range, 0-11) prior lines of therapies, and 37.5% of those previously received ≥2 lines of anti-HER2 therapies. Median exposure time of GQ1001 was 18.5 weeks. The longest treatment duration exceeded 370 days. No DLT was observed in all doses, MTD was not reached up to 8.4 mg/kg, the highest dose tested. Treatment-related adverse events (TRAEs) occurred in 24 subjects (75%). The most common TRAEs (>10.0%) were aspartate aminotransferase (AST) increased (37.5%), thrombocytopenia (28.1%), alanine aminotransferase (ALT) increased (25.0%), pyrexia (21.9%), anemia (18.8%), alkaline phosphatase increased (12.5%), vomiting (12.5%) and nausea (12.5%). Grade ≥3 TRAEs occurred in 9 subjects (28.1%), including 5 myelosuppression, 2 abnormal liver function, 1 hypertension and 1 vomiting. There were no drug-related deaths. The pharmacokinetics analysis showed the concentration of GQ1001 and TAb generally peaked rapidly and declined in a roughly biphasic manner. Among 15 evaluable subjects who received ≥ 7.2 mg/kg, 6 cases achieved confirmed partial response, and 3 had stable disease, the median progression-free survival was 4.8 months

Conclusions: GQ1001 demonstrates an excellent tolerability and promising antitumor activity in heavily pretreated HER2-positive advanced solid tumors, supporting further evaluation of the safety and efficacy of GQ1001 at DRDE of 8.4 mg/kg in the following phase Ib trial. (NCT04450732; sponsored by GeneQuantum Healthcare (Suzhou) Co., Ltd.)

Abstract CT181: A first-in-human (FIH) phase 1 study of SHR-A1921, a TROP-2 targeted antibody-drug conjugate (ADC), in patients with advanced solid tumors

Background: SHR-A1921 is a novel ADC composed of a humanized anti-trophoblast cell-surface antigen 2 (TROP-2) IgG1 monoclonal antibody attached to a DNA topoisomerase I inhibitor via a tetrapeptide-based cleavable linker. Herein, we present the preliminary clinical results of the FIH study of SHR-A1921.

Methods: This is a multi-center, open-label, phase 1 trial (Clinicaltrials.gov, NCT05154604) consisted of dose-escalation, dose-expansion and efficacy-expansion phases. Patients with advanced solid tumors who had failed standard therapy were enrolled. In the dose-escalation phase, SHR-A1921 was planned to be administered from 1.5 mg/kg to 12.0 mg/kg (Q3W, iv) in an i3+3 design, with accelerated titration used for the starting dose; in the dose-expansion phase, additional patients (up to 20-30 per dose level) were enrolled to 2-3 selected tolerable dose levels. The primary objectives were to evaluate the safety and tolerability of SHR-A1921 and to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose.

Results: As of Oct 21. 2022, 38 enrolled patients were included for analysis: 18 enrolled during dose-escalation (1.5 mg/kg, n=1; 3.0 mg/kg, n=4; 4.0 mg/kg, n=8; 6.0 mg/kg, n=5) and 20 during dose-expansion (3.0 mg/kg). 71.1% (27/38) were driver gene-negative non-small cell lung cancer (NSCLC) patients who had previously received platinum-based chemotherapy and anti-PD-(L)1 antibody. 4 patients reported dose-limiting toxicities, with all being grade 3 stomatitis (6.0 mg/kg, n=3; 4.0 mg/kg, n=1). The MTD was established as 4.0 mg/kg. Across all dose cohorts, the most common treatment-related adverse events (TRAEs; ≥30%) were nausea (71.1%), stomatitis (65.8%), anemia (42.1%), vomiting, decreased appetite, decreased weight, and rash (36.8% each). Grade ≥3 TRAEs occurred in 12 patients (31.6%); of these, the most common was stomatitis (n=7, 18.4%), and all other events were reported in ≤2 patients. No patients discontinued study treatment due to TRAEs. As of cut-off date, 10 patients (NSCLC, n=5; triple-negative breast cancer, n=2; ampullary cancer, n=2; ovarian cancer, n=1) had partial response: 4 confirmed and 6 requiring further confirmation. The objective response rate was 33.3% (10/30; 95% CI 17.3-52.8) and disease control rate was 80.0% (24/30; 95% CI 61.4-92.3) in evaluable patients.

Conclusion: SHR-A1921 showed a manageable safety profile and promising anti-tumor activity in patients with pretreated advanced cancer. The trial is ongoing to assess SHR-A1921 at different dosing frequency and the efficacy in selected cancer types.

Abstract CT185: First in human dose-escalation trial with the c-MET targeting antibody-drug conjugate BYON3521

Background: BYON3521 is a novel c-MET targeting antibody-drug conjugate (ADC) with a cleavable linker-duocarmycin (vc-seco-DUBA) payload that causes irreversible alkylation of DNA in tumor cells. BYON3521 has demonstrated potent and selective killing of c-MET expressing tumor cells in preclinical models, even at low c-MET expression levels. In addition, in vitro studies showed that the active toxin can passively permeate the cell membrane and kill neighboring, c-MET negative cells as a bystander effect.

Methods: Patients with previously treated progressive locally advanced or metastatic solid tumors, c-MET positive membrane staining by immunohistochemistry and/or MET-amplified by dual in situ hybridization and/or known activating MET-mutation (excluding exon14m), ECOG performance status 0-1 and adequate organ function are eligible for the dose-escalation part of this first-in-human trial (ClinicalTrials.gov identifier: NCT05323045). An adaptive approach using the Continual Reassessment Method of Neuenschwander (N-CRM model) is used to evaluate the safety of BYON3521 and to determine the maximum tolerated dose (MTD) and recommended dose for expansion (RDE). BYON3521 is administered intravenously every three weeks until tumor progression or unacceptable toxicity.

Results: Up to 01 January 2023, 8 patients (1F, 7M, median age 61 yrs) were enrolled. Tumor types were: 4 colorectal cancer, 1 NSCLC, 1 renal cell carcinoma, 1 esophageal cancer and 1 pancreatic cancer. One patient received 0.8 mg/kg, three patients each 1.6 and 3.2 mg/kg and one patient received 4.8 mg/kg. So far, no grade 3 or 4 related adverse events and no dose-limiting toxicities (DLTs) occurred. Most commonly observed related AEs were decreased appetite/weight, fatigue, abdominal pain, back pain, vomiting and chills. Typical ADC associated AEs as keratitis and pneumonitis have not been observed so far. Stable disease was the best response observed in 2 patients at a dose of 3.2 mg/kg. Human PK was in line with predicted preclinical in vivo PK.

Conclusions: To date, BYON3521 is well-tolerated with no DLTs at the investigated dose levels. Patient enrollment is ongoing and updated safety, efficacy and pharmacokinetic data will be presented. After the dose-escalation phase the trial will continue with expanded cohorts of patients with specific c-MET expressing cancer types.

Abstract CT204: Safety, tolerability, pharmacokinetics, and efficacy of SHR-A1811, an antibody-drug conjugate, in patients with advanced HER2-mutant non-small cell lung cancer (NSCLC): a multicenter, open-label, phase 1/2 study

Introduction: HER2-mutant NSCLC is associated with poor prognosis. SHR-A1811 is a novel antibody-drug conjugate (ADC) consisting of a humanized HER2-directed monoclonal antibody, cleavable tetrapeptide-based linker, and DNA topoisomerase I inhibitor. Here, we report data from the phase 1 portion of a phase 1/2 study with SHR-A1811 in patients with HER2-mutant NSCLC (NCT04818333).

Methods: In this single-arm, dose escalation and expansion phase 1 portion, patients with advanced activating HER2-mutant NSCLC who had failed platinum-based chemotherapy in the advanced or metastatic setting or could not tolerate chemotherapy were enrolled. SHR-A1811 was assessed at doses of 3.2, 4.8, 5.6, 6.4, and 8.0 mg/kg intravenously once every 21-day cycle. Dose escalation and determination of maximum tolerated dose (MTD) was guided by Bayesian logistic regression model with overdose control. Dose selected for expansion was determined based on data from dose-escalation phase. The primary endpoints were safety, MTD and recommended phase 2 dose (RP2D).

Results: At data cutoff (Nov 8, 2022), 50 patients were enrolled. All were stage IV with a median of 3 (range 1-8) prior systemic treatments, including HER2-targeted TKI (66%), immune checkpoint inhibitors (68%), and anti-angiogenic drugs (78%). 96% had a HER2 kinase domain mutation. One patient in 8.0 mg/kg dose cohort had dose-limiting toxicities (grade 4 febrile neutropenia and grade 4 thrombocytopenia). The 4.8 mg/kg dose cohort was expanded to 38 patients. The median follow-up duration was 5.6 months (95% CI 4.2-7.0). 62% of patients remained on treatment. Overall, objective response rate was 40.0% (95% CI 26.4-54.8); median duration of response was 8.3 months (95% CI 5.4-13.7); disease control rate was 86.0% (95% CI 73.3-94.2); median progression-free survival was 10.8 months (95% CI 6.7-15.0). All patients had treatment-related adverse events (TRAEs). 42% of patients experienced grade ≥3 TRAEs, with the most common ones being decreased neutrophil count (30%), decreased white blood cell count (20%), anemia (16%), and thrombocytopenia (12%). Nine patients (18%) had serious AEs deemed related to SHR-A1811. Treatment discontinuation due to AEs was reported in two patients. One death was reported to be treatment related (interstitial lung disease). After single dosing, dose exposure (Cmax and AUClast) of SHR-A1811 increased in a dose-proportional manner. PK parameters of SHR-A1811 and total antibody were similar at all dose levels, with low plasma exposure of free topoisomerase I inhibitor observed.

Conclusion: SHR-A1811 showed tolerable safety profile and durable antitumor activity in heavily pretreated patients with HER2-mutant NSCLC. Dose expansion at 4.8 mg/kg and 5.6 mg/kg is ongoing to establish the RP2D.

Abstract CT213: Biomarker analysis from Phase 1/2 study of tusamitamab ravtansine (SAR408701) in patients with advanced non-small cell lung cancer (NSCLC)

Background Tusamitamab ravtansine is an antibody-drug conjugate of a humanized carcinoembryonic antigen (CEA)-related cell adhesion molecule 5 (CEACAM5)-specific monoclonal antibody linked to DM4. A Phase 1/2 study (NCT02187848) showed tusamitamab ravtansine antitumor activity in pretreated patients (pts) with advanced nonsquamous NSCLC and high CEACAM5 expression. Here, we explore biomarker associations with tumor CEACAM5 expression by immunohistochemistry (IHC), and whether biomarkers predict objective response rate (ORR).

Methods We assessed CEACAM5 expression by IHC, RNA sequencing, and whole exome sequencing (WES) on latest archival tumor samples; and circulating CEACAM5 (cCEACAM5) and CEA (cCEA). We enrolled 2 cohorts of pts with IHC CEACAM5 membrane expression at ≥2+ intensity: in ≥50% of tumor cells (high expressors, HEs, n = 64); and in ≥1% to <50% of tumor cells (moderate expressors, MEs, n = 28). Pts received tusamitamab ravtansine 100 mg/m2 IV every 2 weeks.

Results cCEA and cCEACAM5 were strongly associated (Spearman rho, 0.9), with weak associations between IHC CEACAM5 and cCEA or cCEACAM5 (Spearman rho, 0.3 and 0.4, respectively). Higher levels of CEACAM5 mRNA were observed in CEACAM5 HEs vs MEs (P=0.0027). EGFR and KRAS genetic alterations by WES were present in 44.8% and 65.5% of CEACAM5 HEs, respectively, and 21.4% and 78.6% of CEACAM5 MEs, respectively. Confirmed partial responses were seen in 13/64 HEs (ORR 20.3%) and 2/28 MEs (ORR 7.1%). In CEACAM5 HEs with available baseline (BL) cCEA data, 25/62 (40.3%) had a cCEA level ≥100 µg/L, with a median value of 71.6 µg/L (range 1-8809); corresponding values in CEACAM5 MEs were 7/28 (25.0%) and 12.4 µg/L (range 0.5-684). In response evaluable CEACAM5 HEs with available BL cCEA data (n = 61), ORR was 10/24 (41.7%) in pts with high cCEA (≥100 µg/L) and 3/37 (8.1%) in pts with low cCEA (<100 µg/L); corresponding ORRs in CEACAM5 MEs were 0/7 and 2/21 (9.5%).

Conclusions In CEACAM5 HEs, high cCEA was associated with numerically greater ORR vs low cCEA (41.7% vs 8.1%). Associations were also observed between: cCEA and cCEACAM5; IHC CEACAM5, cCEA, and cCEACAM5; and IHC CEACAM5 and CEACAM5 tumor mRNA levels, but not between IHC CEACAM5 and actionable oncogenic drivers.

Abstract CT214: Preliminary efficacy of telisotuzumab vedotin (Teliso-V) treatment in the 2L/3L setting in MET gene amplified (MET Amp), c-Met protein overexpressing (c-Met OE), non-squamous, non-small cell lung cancer (NSQ NSCLC): Retrospective analysis of LUMINOSITY

Background: Teliso-V is a first-in-class c-Met-directed antibody-drug conjugate comprising the monoclonal antibody telisotuzumab, a cleavable valine-citrulline linker, and the potent microtubule polymerization inhibitor monomethyl auristatin E. In a Phase 2 study (LUMINOSITY, NCT03539536), Teliso-V has shown an acceptable safety profile and encouraging efficacy in c-Met OE NSQ NSCLC. In NSCLC, while c-Met OE is more common than MET Amp, occurring in 25% of patients (pts) versus 1-5%, respectively, ~90% of MET Amp tumors are c-Met OE. We performed a retrospective analysis of LUMINOSITY to characterize the efficacy of Teliso-V in previously treated pts with MET Amp, c-Met OE NSQ NSCLC.

Methods: Pts had locally advanced/metastatic NSQ NSCLC, ≤2 prior lines of systemic therapy, ≤1 line of chemotherapy, MET Amp by fluorescence in situ hybridization (FISH), and tumors that were c-Met OE by central immunohistochemistry (IHC). MET Amp was defined as a ratio of ≥1.8 MET gene copy number (GCN) to chromosome 7 (CEP7) CN by FISH with levels of amplification defined as high, ≥5; intermediate (int), >2.2 - <5; or low, ≥1.8 - ≤2.2. MET GCN by FISH was classified as high, ≥10; int, 5 - <10; or low, <5. c-Met OE was defined as ≥25% of tumor cells at 3+ intensity by IHC; patients were classified as MET IHC 3/25 (25 - 49% tumor cells at 3+) or MET IHC 3/50 (≥50% tumor cells at 3+). Teliso-V was dosed at 1.9 mg/kg IV Q2W.

Results: As of 27 May 2021, 10 pts with MET Amp were treated with Teliso-V. The median age was 69 (48-75), 9 pts were male, 5 were White, 9 had a history of smoking (current or former), and 9 had an ECOG score of 0-1. The median MET/CEP7 ratio was 3.3 (1.84-12.17) with 4, 1, and 5 pts having high, int, and low levels of amplification, respectively. The median MET GCN was 10.98 (6.95-65.00) with 6, 3, and 1 pts having high, int, and low GCN, respectively. 7 pts were MET IHC 3/50 and 3 were MET IHC 3/25. Pts with MET Amp treated with Teliso-V monotherapy had an ORR of 80% (95% CI [44.4, 97.5]; n=8; all partial response) by independent central review, and a disease control rate of 90%. The median duration of response was 6.9 months. Among the 8 responders, 62.5% (n=5/8) are still event-free and being followed up for disease assessment. Teliso-V monotherapy resulted in median progression-free survival (PFS) of 8.0 months (95% CI [1.3, -]; n=10), compared with 5.7 months (95% CI [2.6, 9.8]) on the last prior line of systemic cancer therapy (n=10).

Conclusions: Teliso-V demonstrated a promising ORR in previously treated pts with MET Amp NSQ NSCLC with c-Met OE and improved PFS when compared to last prior systemic cancer therapy. These preliminary data support the ongoing Phase 2 trial of Teliso-V monotherapy in pts with previously untreated MET Amp NSCLC (TeliMET NSCLC-02; NCT05513703), which is currently enrolling.

Abstract CT244: Phase 1/2 study of PRO1184, a novel folate receptor alpha-directed antibody-drug conjugate, in patients with locally advanced and/or metastatic solid tumors

Background: PRO1184 is an antibody-drug conjugate (ADC) directed to folate receptor alpha (FRα), a cell surface antigen overexpressed in multiple cancers including ovarian, endometrial, lung, mesothelioma, and breast cancer. PRO1184 consists of a human monoclonal antibody that selectively binds FRα, a novel cleavable hydrophilic linker, and a topoisomerase 1 inhibitor payload, exatecan. Previous studies demonstrated that the hydrophilic linker confers excellent physicochemical properties and pharmacokinetic (PK) profiles across a range of payload mechanisms and is superior to conventional linkers on these critical parameters for ADCs. In addition, exatecan is broadly active in many tumor types, is membrane permeable, and is not a substrate of multidrug resistance efflux pumps. It may thus lend a robust bystander effect and induce deeper or more durable responses in refractory tumors. Preclinical studies further established that PRO1184 exerts potent antitumor activity in mouse xenograft models with high, moderate, and low FRα expression, consistent with the inherent potency and expected bystander activity of the exatecan payload. PRO1184 is stable in plasma and retains the excellent PK properties and bioactivity of the unconjugated parent antibody. The preliminary safety profile of PRO1184 was more favorable than a benchmarking deruxtecan-based ADC in cynomolgus monkeys. PRO1184 is thus a promising development candidate with a potentially large therapeutic index to benefit a broad population of patients with FRα-expressing solid tumors.

Methods: PRO1184-001 is an ongoing, phase 1/2, open-label dose escalation and expansion study. Eligible patients are adults with metastatic or unresectable solid tumors, including ovarian, endometrial, non-small cell lung, breast cancer, or mesothelioma. Patients must have measurable disease per the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, or mRECIST 1.1 for pleural mesothelioma. Patients must also have previously received therapies known to confer clinical benefit unless considered ineligible, refused by the patient, or not available in the region. PRO1184 is given by intravenous infusion on Day 1 of a 21-day cycle and treatment may continue until disease progression, unacceptable toxicity, or other reason for discontinuation. The primary objectives are to evaluate the safety and tolerability of PRO1184 and to identify the maximum tolerated dose, if reached, and recommended phase 2 dose (RP2D). Part A of the study consists of a dose escalation phase and Part B consists of 4 FRα-expressing tumor-specific expansion cohorts treated at the RP2D. PK, immunogenicity, and antitumor activity will also be evaluated. The study is currently enrolling at sites in the US, with future enrollment in China planned (NCT05579366).

Abstract CT247: A Phase 1/2a, multicenter, open-label, non-randomized first-in-human study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DB-1303 in patients with advanced/metastatic solid tumors

Background: Epidermal growth factor receptor 2 (EGFR2 or HER2) has shown gene amplification/over-expression in more than 30% of all human cancers, including breast cancer, gastric, colon, salivary gland, bladder, and uterine serous carcinoma, and its overexpression in tumors is associated with poor prognosis. DB-1303 (developed by DUALITYBIO INC.) is an antibody-drug conjugate (ADC) comprised of a humanized anti-HER2 IgG1 monoclonal antibody (BAT0606), covalently linked to a proprietary DNA topoisomerase I inhibitor (P1003) via a cleavable linker containing maleimide tetrapeptide (GGFG), with a drug-to-antibody ratio (DAR) of approximately 8. Preclinical studies demonstrated a favorable safety profile and a potent antitumor activity of DB-1303 compared with approved HER2-ADC. These studies warrant further clinical development of the study drug.

Methods: This is a global first-in-human Phase 1/2a study (NCT05150691) to assess the safety, tolerability, and anti-tumor activities of the study drug DB-1303 in patients with pretreated advanced/metastatic solid tumors. Patients should have histologically confirmed HER2-positive or HER2-expressing cancers who failed previously systemic therapies, ECOG 0-1, and adequate organ function. The dose escalation part will evaluate seven ascending dose levels of DB-1303 (accelerated titration design for the first dose level and “3+3” design for the rest dose levels) to determine recommended phase 2 dose (RP2D) or maximum tolerated dose (MTD). The study drug will be administrated iv infusion Q3W, and dose-limiting toxicity (DLT) will be assessed during Cycle 1 (1st 3 weeks). Phase 2a part will initiate after MTD and/or RP2D are determined. Phase 2a part only enrolls the patients with HER2-positive gastric, esophageal, or gastroesophageal junction adenocarcinoma, colorectal cancers, HER2 overexpression and HER2-low endometrial carcinoma, hormone receptor-positive (HR+)/HER2-low breast cancer, HER2-positive breast cancer, and activating HER2-mutated NSCLC. The study treatment of DB-1303 will continue until disease progression, withdrawal of consent, or unacceptable toxicity. Tumor responses will be assessed every 6-9 weeks with RECIST v1.1. The study planned to enroll 253 patients from sites in the United States, Australia, and China (88 patients in Phase I and 165 patients in Phase 2a).

Abstract CT248: First in human trial of DB1305 in patients with advanced malignant solid tumors

Background: Trophoblast cell-surface antigen 2 (Trop-2) is known as tumor-associated calcium signal transducer 2 (TACSTD2) and plays a role in tumor progression, given its active interplay with several key molecular pathways associated with cancer development and progression. DB-1305 is a Trop-2 antibody-drug conjugate (ADC) composed of a humanized anti-Trop-2 immunoglobulin G1 (IgG1) monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor P1021 via a cleavable linker containing maleimide tetrapeptide, with a drug-to-antibody ratio (DAR) of approximately 4. Preclinical in vivo models show that DB-1305 demonstrates robust anti-tumor activity in Trop-2-high triple-negative breast cancer (TNBC), Trop-2-low small cell lung cancer (SCLC) and Trop-2-medium colon cancer but not Trop-2-negative SCLC, demonstrating the dependence of Trop-2 expression for the tumor-suppression activity of DB-1305 and appropriate safety profile supports the intended clinical use. This study aims to evaluate DB-1305 in terms of tolerability and preliminary anti-tumor activity in patients with advanced solid tumors.

Methods: This is an open-label, multicenter, multiple-dose, Phase 1/2a study (NCT05438329), including dose escalation Phase 1 and dose-expansion Phase 2a in patients with pretreated advanced solid tumors. Patients should have histologically documented progressed/refractory disease on or after standard systemic anticancer treatments with proven benefits for their disease or without available standard treatments; adequate performance score (ECOG 0-1); adequate organ function and measurable disease as per RECIST v1.1 while without uncontrolled metastatic central nervous system (CNS) involvement and history of interstitial lung diseases. The dose escalation part will evaluate approximately five ascending dose levels of DB-1305 with accelerated titration for the first dose level followed by a “3+3” design for subsequent dose levels to identify the optimal dose, which will enroll up to 70 patients from the United States and China. Upon determining the optimal dose, seven tumor cohorts of 10-40 patients will be included in the dose expansion part, including non-small cell lung cancer, SCLC, hormone receptor-positive breast cancer, and TNBC. DB-1305 will be dosed in both parts until disease progression, clinical deterioration, withdrawal of consent, or unacceptable toxicity. The primary objectives are evaluating safety tolerability and identifying the optimal dose from Phase 1; assessing safety, tolerability, and objective response rate per RECIST v1.1 from Phase 2a. Secondary objectives include the assessment of the pharmacokinetics and immunogenicity of DB-1305. Exploratory objectives include the assessment of pharmacodynamic biomarkers and Exposure-Response correlation. As of 19 Dec 2022, 20 patients have been enrolled in the dose escalation part.

Abstract CT255: ELU-FRα-1: a study to evaluate ELU001 in patients with solid tumors that overexpress folate receptor alpha (FRα)

Background: ELU001 is a novel, first-in-class, new molecular entity described as a C’Dot Drug Conjugate (CDC). ELU001 consists of ~13 folic acid targeting moieties and a payload of ~22 molecules of the topoisomerase-1 inhibitor, exatecan. Folic acid and exatecan are covalently bound by non-cleavable and cathepsin-B cleavable linkers, respectively, to short polyethylene glycol chains which surround the C’Dot’s silica core. CDCs are very small (~6 nm), allowing greater ability to penetrate more efficiently into solid tumors compared to ADCs. CDCs are rapidly eliminated by the kidneys, which is expected to lead to less toxicity than ADCs that have a longer half-life in circulation. ELU001’s high avidity is designed to promote binding to FRα on the surface on FRα overexpressing cancer cells with a wide range of antigen expression including high, moderate and low expressing tumor cells. Following antigen binding, ELU001 internalizes into the tumor cell, and traffics to the lysosome where enzymatic cleavage releases the exatecan payload. The first-in-human trial, ELU-FRα-1, is currently recruiting patients that have advanced, recurrent or refractory tumors associated with indications that are known to potentially overexpress FRα and have been shown to be topoisomerase 1 inhibitor-sensitive, and, in the opinion of the Investigator, have no satisfactory therapeutic options available.

Methods: This is a Phase 1/2 multicenter, open label clinical trial with two parts: Part 1 Dose Escalation and Part 2 Tumor Group Expansion Cohort(s). Part 1 is a basket trial enrolling patients with cancer types with a high likelihood of having FRα overexpressing tumors, (specifically, ovarian, endometrial, colorectal, gastric, gastroesophageal junction, triple negative breast, or non-small cell lung cancers, or cholangiocarcinoma). Patients are receiving ELU001 weekly (QW) (once a week for 3 weeks, 1 week rest), every other week (Q2W, with no rest between cycles), or every three weeks (Q3W). Analysis of FRα expression will be retrospectively determined. Part 2 will use Simon’s Two-Stage design to evaluate 4-6 Expansion Cohorts, comprised of tumor histologies anticipated to have greatest activity to ELU001 treatment. FRα will be prospectively determined. The primary objective for Part 1 is to identify the MTD/RP2D. The primary objective for Part 2 is to determine the ORR. Secondary objectives include DOR, PFS, TFST, PFS2, OS, frequency, severity and tolerability of adverse events, PK, ADA, and FRα expression assessments. Part 1 Dose Escalation will recruit about 25 patients per dose regimen (QW; Q2W; Q3W). The first stage of Part 2 (Dose Expansion) will recruit about 15 patients per tumor group expansion cohort. The study is actively enrolling in the US and currently recruiting in Q2W Cohort 201 and Q3W Cohort A. QW Cohorts 1-3 and Q2W Cohort 101 are complete. Clinical trial information: NCT05001282.

Abstract CT268: Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial

Despite provocative preclinical results, dose-limiting toxicities have precluded rational combinations of cytotoxic chemotherapies that increase DNA damage with DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted delivery of cytotoxic chemotherapy might enable tolerable and active combinations with DDR inhibitors. We conducted a phase I clinical trial combining ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib with sacituzumab govitecan, a trophoblast cell surface antigen 2 (Trop-2) directed antibody drug conjugate (ADC) that delivers high tumoral concentrations of topoisomerase 1 (TOP1) inhibitor SN-38. Depletion of ATR, the main transducer of replication stress is synthetically lethal with double-strand breaks (DSB) generated by TOP1 inhibitors. Patients with DDR gene-mutated or high replication stress solid tumors were enrolled since such tumors are particularly susceptible to ATR inhibition. Primary end point was identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Using 3 + 3 dose escalation, sacituzumab govitecan (8-10 mg/m2, days 1, 8) and berzosertib (140-210 mg/m2, days 2, 9) were administered to 12 patients across three dose levels in 21-day cycles. The combination was well tolerated, with improved safety profile over conventional chemotherapy-based combinations, which allowed dose escalation to the highest planned dose level. There were no dose limiting toxicities. Common treatment-related adverse events (TRAE) were neutropenia (41.7%), diarrhea (50%), and fatigue (50%). Grade 3 TRAEs occurred in 58.3% of patients and included neutropenia (25%) and diarrhea (8.3%). There were no instances of febrile neutropenia or clinically significant grade 4 TRAEs. Pharmacodynamic studies showed evidence of ATR inhibition and enhanced DNA DSB in response to the combination. While no tumor responses were seen in three patients with DDR defects including BRCA1 and ATM mutations, two patients with neuroendocrine prostate cancer, a highly aggressive subtype of prostate cancer, showed partial or metabolic responses. A patient with EGFR-transformed small cell lung cancer (SCLC) also experienced partial response, together yielding objective responses in 3 of 12 evaluable patients (25%). Ongoing phase II expansion cohorts are evaluating efficacy of sacituzumab govitecan 10mg/m2 and berzosertib 210mg/m2 in patients with SCLC, extra-pulmonary small cell cancers, and DDR-mutated solid tumors. ADC-based delivery of cytotoxic payload represents a new therapeutic paradigm to extend the benefit of DDR inhibitors to target replication stress and chemotherapy resistance, with minimal added toxicities. Clinical trial information: NCT04826341

Abstract LB011: Development of novel protein drug conjugates (PDCs) for the selective targeting of ALPP/ALPPL2 expressing tumors

The placental alkaline phosphatases, ALPP and ALPPL2, are glycosylphosphatidylinositol (GPI)-anchored cell-surface proteins that are expressed in the placenta during fetal development but have very little expression on normal adult tissue. These proteins are over-expressed in a number of different solid tumor indications including ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers. As a result of the highly restricted normal tissue expression and over-expression in cancer, ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. The recent initiation of a Phase I clinical trial utilizing a full-length antibody ADC has fueled further interest in these oncology targets. Small protein domain binders, which have the capacity to penetrate deeper into solid tumors and can be engineered into multiple therapeutic formats in a modular fashion, offer a number of potential benefits over full-length antibodies as the targeting vehicle in PDC therapeutics. We report the identification and characterization of a series of high affinity ALPP/ALPPL2 specific single domain VHH binders which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Through application of the Almac Discovery PDC technology platform, these VHH domains have been reformatted into a suite of homogenous site-specifically labelled drug conjugates, with defined drug to antibody ratios, in high yields. These conjugates, which are based on mono- and bi-paratopic Fc fusion formats, employ both clinically established and novel linker-toxin combinations. In pre-clinical studies, the lead bi-paratopic PDCs were well tolerated in vivo and showed excellent anti-tumor efficacy in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric and pancreatic carcinoma, with sustained regressions still observed 10 weeks after administration of the final dose of agent. These agents have physicochemical and pharmaceutical properties suitable for further development and we anticipate that the excellent pre-clinical efficacy profile of lead PDCs will translate to a highly differentiated product for the treatment of a variety of solid tumor indications.

Abstract LB025: First disclosure of AZD5335, a TOP1i-ADC targeting low and high FRα-expressing ovarian cancer with superior preclinical activity vs FRα-MTI ADC

Folate receptor alpha (FRα) is a cell surface GPI-anchored protein overexpressed in several solid tumors with highest prevalence in ovarian cancer and lung adenocarcinoma but restricted expression in normal tissues. An antibody drug conjugate (ADC) with a microtubule inhibitor (MTI) payload recently received accelerated approval from the FDA for FRα-expressing platinum-resistant ovarian cancer. We describe for the first time the preclinical activity of AZD5335, an FRα-targeting antibody conjugated to AZ’s proprietary topoisomerase 1 inhibitor (TOP1i) payload, AZ14170132, with a homogeneous drug-to-antibody ratio of 8 (DAR8) and potential benefits vs an MTI-based ADC. AZD5335’s primary mechanism of action is to deliver TOP1i payload into FRα-expressing cancer cells, leading to DNA damage and apoptotic cell death. The TOP1i payload mediates bystander killing, which is important for targeting tumors with less than uniformly positive expression. Here, we report that a single dose of AZD5335 at 2.5 mg/kg was sufficient to provide a robust and durable anti-tumor response in FRα-expressing ovarian cancer cell line xenografts (CDX) with a tumor growth inhibition (TGI) of 75%-94% and median best tumor volume reduction >30% in 14/17 (82%) ovarian cancer patient-derived xenografts (PDX) evaluated. FRα-expression levels (by IHC and deep-learning based image analysis) correlated with efficacy in the tested PDX models, and we observed that AZD5335 was also active in models with low levels of target expression (75% of cells with FRα staining of 2+), expected to be representative of patients who would be ineligible for treatment with the MTI-ADC. Furthermore, AZD5335 demonstrated superior activity vs an FRα-MTI benchmark ADC with respect to anti-tumor activity and duration of response in two PDX models with low-to-medium FRα expression at equal or higher drug doses (e.g., in OV0857-CIS: 96% TGI vs 24% TGI at 5 mg/kg and 95% TGI vs 2% TGI at 2.5 mg/kg of a single IV dose AZD5335 and FRα-MTI, respectively). These data indicate that AZD5335 is a promising therapeutic candidate for the treatment of ovarian cancers across the spectrum of FRα-expression.

Abstract LB028: Reprogramming of tumor-infiltrating immune cells using a tumor-associated macrophage-targeted TLR7 agonist

Myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are two of the more immunosuppressive cell types in a tumor microenvironment (TME). Although TAMs and MDSCs can be converted from tumor-supporting to tumor-suppressing phenotypes by treatment with proinflammatory immune stimulants (e.g. TLR7 agonists) in vitro, such strategies have proven to be too toxic in vivo because of their systemic activation of immune cells. Therefore, for such immune stimulants to be used to reprogram the immune system in solid tumors, they must be targeted specifically to the immune cells in the TME.

One strategy for targeting TAMs/MDSCs is to exploit a receptor that is expressed solely on these cell types. Because folate receptor beta (FRβ) is expressed exclusively on myeloid cells and since FRβ acquires its ability to bind folic acid (FA) only in inflamed tissues, we have previously exploited FA to target attached TLR7 agonists (TLR7a) to FRβ+ TAMs/MDSCs in solid tumors. While significant repolarization of TAMs and MDSCs was observed in this previous study, premature systemic release of the attached TLR7a caused unwanted toxicity. However, since FRβ was later found to reside in the same endosome as TLR7, we hypothesized that constructing a more stable FA-TLR7a conjugate with a non-cleavable linker should not only prevent premature TLR7a release, but also deposit the FA-TLR7a into an endosome containing a TLR7 receptor, thereby enabling selective activation of TAMs and MDSCs in the TME. The data summarized here confirm this hypothesis. Thus, free TLR7-1A was found to stimulate massive production of IL-6, TNF-α and IFNγ upon addition to human peripheral blood in vitro or injected into live mice, suggesting cells in both species respond aggressively to TLR7-1A. However, performance of the same studies with FA-targeted TLR7-1A stimulated barely or nondetectable levels of the inflammatory cytokines, confirming that FA-TLR7-1A is unable to enter and activate cells that lack a folate receptor (FRβ). More importantly, intravenous administration of FA-TLR7-1A into mice bearing an orthotopic 4T1 breast tumor totally blocked metastases and induced tumor regression without causing detectable toxicity. Flow cytometric analyses of the residual tumor tissue further demonstrated that FA-TLR7-1A increased M1/M2 macrophage ratios and elevated infiltration of CD8+ T cells into the tumor masses. Taken together, these data demonstrate that a targeted TLR7 agonist can rejuvenate immune cells in a solid tumor without systemically activating the immune system.

Abstract LB030: SHR-A1921, a novel TROP-2 ADC with an optimized design and well-balanced profile between efficacy and safety

Trop-2 is a promising target for ADC therapy due to its high expression in many solid tumors. The approval of Trodelvy, a Trop-2 directed ADC, for the treatment of refractory or drug-resistant triple negative breast cancer (TNBC) demonstrated the therapeutic value of Trop-2-targeted ADC. However, a Boxed Warning for severe or life-threatening neutropenia and severe diarrhea suggests the safety of Trodelvy needs to be improved. Here, we presented a novel Trop2-directed ADC, SHR-A1921, consisting of a topoisomerase I inhibitor (Proprietary payload, SHR9265) conjugated to a proprietary IgG1 mAb via cleavable linkers. SHR-A1921 demonstrated several advantages over other Trop-2 directed ADCs in the field. SHR9265 is a novel exatecan derivative designed by Hengrui with a better liposolubility and cellular permeability. SHR-A1921 had a drug-to-antibody ratio (DAR) of 4. Compared with other Trop-2-targeted ADCs in the field, such as Trodelvy, TINA-SHR79711 (a molecule synthesized using the published structure of DS-1062), and SKB264, SHR-A1921 has considerable advantages as follows: (1) Stronger binding affinity to both human and rhesus macaque TROP-2 than TINA-SHR79711; (2) Improved plasma stability in plasma of different species presumably due to the proper steric hindrance which was purposely designed on the payload for reducing non-intended cleavage; (3) Stronger bystander cell killing effect presumably due to the increased lipophilicity of the payload vs. that of the payload in TINA-SHR79711; (4) Superior in vivo efficacy in a PSCC2 CDX Model (FaDu) with high Trop-2 expression (TGI 101% vs 53% [TINA-SHR79711] @ 1 mpk) and in an ovarian cancer CDX Model (SK-OV-3) with moderate Trop-2 expression (TGI 63% vs. 23% [TINA-SHR79711] @ 3 mpk; 87% vs. 16% [TINA-SHR79711] @ 10 mpk); (5) ≥ 2X longer half-life in patients# vs. SKB264 vs. IMMU-132, supporting more flexible dosing frequency; (6) Lower free toxin/ADC ratio# regarding PK exposure in patients compared with SKB264 (< 1% vs. 5-6%); (7) approximately linear pharmacokinetics profile in patients with T1/2 ranging from 2.5 to 4.5 days. In summary, SHR-A1921 is a novel anti-TROP2-targeted ADC with a high permeable payload and optimized DAR demonstrating great stability and high potency in both in vitro and in vivo studies. SHR-A1921 also showed compelling efficacy and good safety profile from 50+ subjects of Phase I clinical trial in China (NCT05154604). Pivotal phase III trial for NSCLC is planned in China. (Notes: 1. TINA-SHR7971 is a molecule that Hengrui synthesized using the published structure of DS-1062. 2. PSCC: pharyngeal squamous cell carcinoma. #. non-head-to-head comparison.)

Abstract LB031: SHR-A1811, a novel anti-HER2 ADC with superior bystander effect, optimal DAR and favorable safety profiles

Current clinical HER2-targeting ADCs, especially T-DXd, have shown strong efficacy in HER2-expressing/mutant cancers, however, with adverse events such as hematologic toxicities and interstitial lung disease (ILD)/pneumonitis. Here we presented a potential best-in-class HER2-directed ADC, SHR-A1811, which was composed of trastuzumab, a stable and cleavable linker, and a novel topoisomerase I inhibitor payload (SHR9265). SHR9265 was a delicately selected exatecan derivative with a better liposolubility and cellular permeability: SHR9265 had a higher AlogP value than SHR7971, synthesized using published DXd structure (3.67 vs. 2.72). Consistently, SHR9265 showed ~5 times higher membrane permeability (PAMPA model) at pH5 and pH7.4. Cell killing activity of SHR9265 was ~3 times more potent than SHR7971. SHR-A1811 had a drug-to-antibody ratio (DAR) of 5.7, and showed HER2-dependent growth inhibition against various breast cancer and gastric cancer cell lines. In the bystander killing system, SHR-A1811 was able to kill both SK-BR-3 (HER2+) and MDA-MB-468 (HER2-) cells when co-cultured, with the IC50 on MDA-MB-468 of 0.28 nM. The in vitro efficacy of SHR-A1811 was comparable with anti-Her2-SHR9265 (DAR 7.5) and ADC1 (synthesized using T-DXd structure), and stronger than anti-Her2-SHR9265 (DAR 3.5). In SK-BR-3 (HER2 high), JIMT-1 (HER2 moderate) and capan-1 (HER2 low) xenograft models, SHR-A1811 treatment resulted in a dramatic and sustained inhibition of tumor growth. A significantly stronger antitumor activity was observed for SHR-A1811 than ADC1 under the same dosages. Moreover, SHR-A1811 showed good stability and improved safety profiles, presumably due to the proper steric hindrance which was purposely designed on the payload (SHR9265). Less than 2% of payload release was observed in human plasma after a 21-day incubation. The HNSTD of SHR-A1811 in cynomolgus monkeys was 40mpk with thymus as the main target organ. No death and lung lesions were observed at dose levels up to 70mpk for 42 days, while ADC1 led to one male cynomolgus monkey death on day 10 at 70mpk with multiple lung damages. These findings were consistent with safety profiles observed in a multi-center, dose-escalation phase I clinical trial (NCT0444620). Patients with different solid tumor types received initial intravenous doses of SHR-A1811 from 1 to 8.0 mg/kg. Only trace amount of free toxin was detected. The Cmax of payload was 3.85 ng/ml at 8mg/kg of SHR-A1811. The incidence of G2 ILD was < 2.5% and the treatment discontinuation rate was 5.1% across doses. In summary, with a highly permeable payload, optimized DAR, great potency and better clinical safety profiles, SHR-A1811 has demonstrated the best-in-class potential. Currently SHR-A1811 has entered phase II and phase III clinical studies for breast cancer, gastric cancer, colorectal cancer, and NSCLC (NCT05424835, NCT05482568, NCT04818333, NCT05349409).

Abstract LB042: Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd)in tamoxifen-resistant ER+ breast cancer cells by inducing DNA damage and apoptosis

Background: HER3, a member of the ERBB family of receptor tyrosine kinases that activates multiple oncogenic signaling pathways, is overexpressed in 50-70% of breast cancers (BC). HER3 mRNA expression is highest in luminal (ER+) breast tumors. Approximately 30% of ER+ breast tumors are de novo resistant to tamoxifen. Therefore, therapeutically targeting HER3 with HER3-DXd, an antibody-drug conjugate (ADC) composed of a fully human anti-HER3 IgG1 monoclonal antibody (patritumab) covalently linked to a topoisomerase I (TOP I) inhibitor payload (deruxtecan) via a tetrapeptide-based cleavable linker, can be an effective treatment for tamoxifen-resistant (TMR) ER+ BC. After assessing HER3-DXd’s efficacy as a single agent, we sought to identify a synergistic partner to maximize its antitumor activity in HER3+/ER+ TMR BC.

Methods: Whole-genome high-throughput siRNA screening (Ambion Silencer Select Human Genome siRNA Library V4) was performed to identify synergistic partners for maximizing HER3-DXd’s antitumor efficacy. The synergistic antitumor effects were assessed in vitro using a soft agar colony formation assay and a clonogenic assay in TMR HER3+/ER+ MCF7 and T47D BC cells and in vivo using xenograft mouse models of these cells. Targeting specificity was determined using siRNA. Treatment effects on cell cycle progression, DNA damage, apoptosis, and expression of proteins of interest were assessed by flow cytometry, comet assay, staining with annexin V-PE and 7-AAD, and Western blotting, respectively.

Results: HER3-DXd inhibited the anchorage-independent growth of HER3+/ER+ cells by >50% at 5 nM and their colony formation at 5-25 nM (P < 0.05). Among the synergistic targets identified by whole-genome high-throughput siRNA screening, inhibiting ATR with siRNA or BAY1895344 showed the greatest synergistic effect with HER3-DXd in TMR HER3+/ER+ BC cells. In contrast, no synergistic effect was observed with the combination of BAY1895344 plus patritumab or control ADC (IgG-DXd), suggesting its dependence on HER3-DXd-mediated delivery of DXd. To further confirm the targeting specificity, we knocked down ATR or TOP I expression using siRNA in TMR HER3+/ER+ BC cells and then treated the cells with HER3-DXd or BAY1895344, respectively. A synergy was also observed, indicating that the drugs achieve the synergy by targeting ATR and TOP I. The combination of HER3-DXd plus BAY1895344 reprogrammed cell cycle progression from G2/M arrest to sub-G1 arrest by inhibiting both ATR/Chk1/cyclin A2/CDK2 and ATR/Chk1/cyclin E/CDK2 signaling. The combination also induced DNA damage, which was further confirmed by the reduced expression of H2AX, an ATR substrate that contributes to DNA repair, and the increased expression of γH2AX (phospho-H2AX at Ser139), an indicator of DNA damage. HER3-DXd and BAY1895344 synergistically inhibited the growth of both TMR HER3+/ER+ MCF7 (P < 0.0001) and T47D (P < 0.01) xenografts in mice.

Conclusion: The combination of HER3-DXd plus ATR inhibitors has therapeutic potential for overcoming tamoxifen resistance in HER3+/ER+ BC.

Abstract LB209: An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells

Introduction: Multiple myeloma (MM) is a neoplastic malignancy characterized by the abnormal proliferation of plasma cells with excessive antibody production in the bone marrow. It accounts for approximately 1.8% of all new cancer cases and 2.1% of cancer deaths in the United States and occurs most frequently among older adults (age > 75). While there are a number of treatments with high response rates, MM relapses frequently within a few years and is considered incurable. Herein, experimental studies and results of a new therapeutic agent under development, TE-1146, are reported. TE-1146 is an antibody drug conjugate (ADC) composed of two therapeutic agents already in use in MM, namely, an anti-CD38 antibody, daratumumab (Dara), and lenalidomide (Lena).

Method: TE-1146 is designed by employing “HIDAR” technology platform, which enables the preparation of homogeneous ADCs with high DAR (drug to antibody ratio). In the TE-1146 molecules, the Fab is replaced by scFv and a cysteine-containing Zn2+-binding motif is engineered at the C-termini of the H chains. Lena molecules are assembled into drug bundles containing 3 Lena molecules and a maleimide group. Two drug bundles are conjugated site-specifically to the Zn2+-activated SH groups of the reconfigured antibody molecule, creating an ADC with DAR of 6. TE-1146 was investigated for cytolytic effects against human MM cell lines H929 in cell cultures in vitro and transplanted H929 tumor in NOD-SCID mice in vivo, in comparison with Dara, Lena, and their combination.

Results: In human plasma, TE-1146 has stability comparable with Dara, and the Lena molecules in the drug bundles remain conjugated. In cultures of H929 cells, TE-1146 caused the lysis of H929 cells at least 100 times more effectively than Dara, Lena, and Dara/Lena combination, based on IC50 comparison. It was shown that H929 cells internalized and degraded TE-1146 and freed Lena. In NOD-SCID mice, the subcutaneously transplanted H929 cells were allowed to grow into solid tumors in 14 days, and TE-1146 and other agents for comparison were administered intraperitoneally. It was found that one single dose of TE-1146 at 20 nmol/kg (conjugated with Lena at 120 nmol/kg) could retard the growth of the transplanted tumor and ultimately eliminate the tumor over 28-42 days, while the combination of one dose Dara at 20-80 nmol/kg and Lena of 46 μmol/kg given daily could not eliminate the tumor. The amount of Lena used in the combination treatment over a 28-day course is 10,700 times that of Lena in TE-1146.

Conclusion: Lenalidomide is extremely powerful in killing multiple myeloma cells if brought into the MM cells. It is estimated that a very minute amount, probably less than 0.01%, of intraperitoneally injected lenalidomide gets into the transplanted MM tumor in the mouse model. TE-1146 may be a more effective and less toxic drug than Daratumumab/Lenalidomide combination in treating patients with hard-to-treat MM.

Abstract LB212: BCG022: A novel bispecific antibody-drug conjugate targeting HER3 and MET

HER3 is a unique EGFR family member that plays a role in both tumor progression and drug resistance. Its expression can act as a bypass mechanism for EGFR and HER2-targeted therapies, resulting in therapeutic resistance. MET has also been reported as a bypass resistance mechanism to EGFR-TKI treatment. HER3 and MET are co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both proteins may provide clinical benefit. We generated fully human bispecific antibodies (bsAbs) targeting HER3 and MET with cross-species reactivity, using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate future bispecific antibody assembly. These 1+1 bsAbs have demonstrated enhanced internalization compared to the parental monoclonal antibodies in multiple cancer cell lines. These bsAbs were then conjugated with Monomethyl auristatin E (MMAE) to generate HER3 and MET-targeting bispecific ADC (BCG022) candidates. In vivo drug efficacies are being screened using cell-derived hepatocellular carcinoma (HCC) and gastric carcinoma xenografts, as well as patient-derived gastric and pancreatic xenograft models. Collectively, these results suggest that BCG022 has the potential to be a novel therapeutic option for HER3 and MET co-expressing tumors.

Abstract LB213: Identification of DM004, a first-in-class anti-5T4/MET bispecific antibody-drug conjugate

Aberrant MET signaling is frequently found in various types of solid tumors, and is correlated with oncogenic transformation, treatment resistance and poor prognosis. While MET remains an attractive therapeutic target, it is widely expressed on the surface of epithelial and endothelial cells, including normal tissues and tumors. To date, MET-targeting agents are associated with adverse clinical effects, including hypoalbuminemia, peripheral edema and pneumonitis, indicating that alternate treatments and/or modalities are needed. Intriguingly, MET antigen is commonly co-expressed with the oncofetal antigen 5T4 in various cancer types, including head and neck, lung and pancreatic cancer. While 5T4 is highly expressed on primary and metastatic cancers and is associated with adverse clinical outcomes in solid tumors, expression on normal adult tissues is very limited. Although several therapeutic agents targeting 5T4 antigen are currently being evaluated in human clinical studies, none have yet entered the market. To address these challenges, we hypothesized that targeting both MET and 5T4 with a bispecific antibody-drug conjugate (BsADC) could provide a more targeted therapeutic strategy to effectively eliminate tumor cells and reduce systemic toxicity. Here, we report that we have successfully generated two bispecific antibody candidates targeting both 5T4 and MET. The candidates were conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker to generate DM004 BsADCs, i.e., Top1-MMAE and Top2-MMAE. In vitro, DM004 BsAbs demonstrated enhanced internalization in the NCI-H226 cell line compared to its parental monoclonal and monovalent anti-5T4 and anti-MET antibodies. In vivo, DM004 BsADCs exhibited robust anti-tumor activity in cell line-derived and patient-derived xenografts of gastric cancer and lung cancer, respectively. In particular, DM004 Top2-MMAE outperformed benchmark ADCs in lung BP0508 PDX models. In summary, we have identified a novel BsADC which may be a promising future treatment for cancers co-expressing 5T4 and MET.

Abstract LB214: A first-in-class bispecific antibody-drug conjugate (DM002) targeting HER3 and the juxtamembrane domain of MUC1

Despite the entry of new anti-cancer drugs into the market, there were more than 10 million deaths from cancer globally in 2021, with cancers of the lung, stomach, breast, and pancreas contributing to the most cancer-related deaths in China and the USA. Accordingly, there is an urgent need for improved therapeutic interventions. Antibody-drug conjugates (ADC) are novel drugs that exploit the specificity of a monoclonal antibody for target antigens expressed on cancer cells in order to achieve targeted delivery of a potent cytotoxic payload. More recently, bispecific ADCs (BsADC) targeting two tumor-associated antigens (TAA) have been developed to further amplify tumor cell specificity while minimizing toxicity to normal tissue, thus allowing for broader therapeutic windows. HER3 and MUC1 are two TAAs that are commonly expressed/co-expressed on multiple tumor types, including lung, gastric, breast and pancreatic cancer; however, neither antigen has been successfully targeted by effective drugs: of the candidates targeting HER3 antigen being evaluated in human clinical studies, most are well-tolerated, but with limited efficacy. On the other hand, high levels of MUC1 in diseased tissues can undergo auto-proteolysis, so that drugs targeting the MUC1-N region will be neutralized before reaching the tumor tissues, thereby limiting the recognition of tumor cells. To overcome these challenges, we generated fully human bispecific antibody candidates with human-monkey cross-species reactivity that target HER3 and the juxtamembrane domain of MUC1. These bsAbs exhibit higher endocytic activity in HER3-low tumor cell lines compared with other currently available HER3 mAb. These bsAbs were subsequently conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker to obtain first-in-class BsADC candidates, DM002. DM002 candidates showed robust anti-tumor activity in multiple CDX and PDX models of lung, breast, gastric and pancreatic cancer; most notably, DM002 candidates outperformed benchmark ADCs in BP0508 lung PDX models. Together, these data indicate that DM002 will be a promising therapeutic drug for patients with HER3 and MUC1 co-expressing tumors.

Abstract LB215: A first-in-class anti-TROP2/EGFR bispecific antibody-drug conjugate, DM001, exhibits potent anti-tumor efficacy

EGFR is a well-established target for the treatment of many cancers. However, limitations encountered with current therapies, such as drug resistance and low cytotoxicity, indicate a need for alternative treatments. In particular, antibody-drug conjugates (ADCs) are a promising new therapeutic strategy, due to their potent killing effects and high target specificity. However, the toxicity of the payload can often cause safety concerns with ADCs, so their efficacy and safety must be carefully evaluated. With these challenges in mind, we hypothesized that development of a bispecific ADC (BsADC) targeting EGFR and a second tumor-associated antigen could help to improve the tumor selectivity of the ADC, thereby limiting the occurrence of on-target off-tumor effects. TROP2 and EGFR are co-expressed in multiple types of solid tumors, including head and neck, esophageal, lung, and pancreatic cancers, indicating that this target combination could provide therapeutic benefit for a wide range of tumors. Herein, we developed a novel bispecific ADC, DM001, targeting TROP2 and EGFR, conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker. In vitro, internalization of DM001 bsAb into a TROP2+EGFR+ cell line is comparable with that of its parental monoclonal anti-TROP2 or anti-EGFR antibodies. Tumor killing of double positive cell lines is also comparable between DM001 and its parental ADCs. Compared with single positive cells, DM001 can selectively bind and better kill double positive cells. Mechanistically, DM001 delays progression of the cell cycle and increases the frequency of apoptosis in vitro in an antigen-dependent manner. Pharmacokinetic analyses in mice with humanized FcRn (B-hFcRn) demonstrated a similar half-life of DM001 to isotype controls. Importantly, DM001 demonstrated strong anti-tumor activity in several cell line-derived and patient-derived xenografts, including lung and pancreatic tumors. Notably, the efficacy of DM001 was superior to benchmark ADCs in A431 and Panc.02.03 xenografts. Interestingly, the efficacy of DM001 was superior to its parental ADCs in BP0508 lung cancer and BP0209 pancreatic cancer PDX models, but not obvious in Panc.02.03 CDX models, indicating that DM001 may effectively target heterogeneous tumors, which better mimic the tumor microenvironment in patients. In summary, DM001 is a novel bispecific ADC with promising therapeutic potential that can be further exploited to treat TROP2 and EGFR co-expressing tumors.

Abstract LB216: Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer

Triple-negative breast cancer (TNBC) accounts for 15-20% of metastatic breast cancer incidence, and remains an area of unmet clinical need due to the low rates of overall survival. Recently, the TROP2-targeting ADC sacituzumab govitecan has received an accelerated approval from the FDA for adult patients with metastatic TNBC, as more than 85% of TNBC is marked by TROP2 overexpression. However, the clinical efficacy of ADC therapies targeting TROP2 alone is limited by its on-target toxicity. In an effort to offer therapeutic alternatives that limit this toxicity, we sought to identify other targets to combat metastatic TNBC in combination with TROP2. PTK7 is highly expressed in breast cancer; notably, PTK7 expression is higher in TNBC than non-TNBC, and is correlated with worse prognosis, tumor metastasis and TNBC progression. PTK7 has also been demonstrated to be enriched in tumor-initiating cells (TICs) in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). We generated fully human anti-human PTK7 x TROP2 bispecific antibodies (bsAbs) from RenLite® mice, which harbor the complete human heavy chain immunoglobulin variable domain with a common human kappa light chain for subsequent bispecific antibody assembly. These bsAbs demonstrated reactivity to human, monkey, and dog antigens, and showed enhanced internalization in vitro compared with parental PTK7 antibodies. In addition, these bsAbs showed favorable tumor cell selectivity, as there was minimal internalization of the monovalent antibodies. These bsAbs were than conjugated with Monomethyl auristatin E (MMAE) to generate anti-PTK7 x TROP2 bispecific ADC (BCG033) candidates. BCG033 candidates showed potent anti-tumor activity in several cell line-derived xenografts including TNBC xenografts, indicating that BCG033 has strong therapeutic potential in TNBC and other PTK7/TROP2 co-expressing cancers. Patient-derived TNBC xenografts with co-expression of PTK7 and TROP2 have been screened for future in vivo drug efficacy screening. In summary, BCG033 has the potential to exert anti-tumor efficacy in TNBC and other solid tumors co-expressing PTK7 and TROP2.

Abstract LB218: Developing a bispecific anti-ROR1 antibody drug conjugate for hematological and solid tumor treatment

Developing a bispecific anti-ROR1 Antibody Drug Conjugate for hematological and solid tumor treatment

Receptor tyrosine kinase ROR1 is a type I transmembrane protein belongs to the ROR family members. ROR1 is a receptor for Wnt family signaling molecules Wnt5a and is a key regulator of normal cellular process, including cell proliferation, survival, and migration. It is also involved in the development and progression of many types of cancer. Although being an oncofetal protein with limited expression in most of the normal tissues, ROR1 is expressed abnormally in various hematological and solid cancers, making it a highly attractive target for antibody-drug conjugate (ADC) therapy. The current clinical results of ROR1 ADC have been promising in treating patients with relapsed and/or refractory (R/R) hematologic malignancies.

Utilizing our unique and innovated linker platform, we screened many anti-ROR1 ADCs, with defined DAR=4. Those unique ADCs consist of a humanized monoclonal antibody (mAb against single epitope) or a bispecific antibody (BsAb against two epitopes), stably conjugated to an antimitotic agent. The bispecific mAbs that target to two different epitopes of ROR1, are superior to those antibodies that target to single epitope in the binding to ROR1-expressing tumor cells, the induction of tumor cell death and anti-tumor immunity. Our novel linker structure prevents payloads from coming off of the antibody during the circulation, significantly reduced the off-target toxicity. In vitro and In vivo studies demonstrated the antitumor activity of anti-ROR1 ADCs outperformed the lead anti-ROR1 ADC currently in phase II/IIl trial, providing a promising treatment for hematological and solid cancers with a better safety profile and a larger therapeutic window. The lead candidate molecule, BR111A will start the preclinical studies soon.

Abstract LB219: Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation

The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using a Topoisomerase 1 (Topo1) inhibitor as payload with highly favorable biophysical properties and superior anti-tumor efficacy compared to Trastuzumab deruxtecan in head-to-head in vitro and in vivo studies. Based on trastuzumab as the targeting antibody and a Topoisimerase 1 inhibitor as payload, we generated highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of 2. In in-vitro assays on target positive cell-lines, the Araris Topo 1 ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved Trastuzumab deruxtecan which has a DAR of 8. Moreover, the ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while Trastuzumab deruxtecan showed significant payload loss during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for payload release was highly efficient in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile similar to the unmodified trastuzumab parent antibody. In efficacy studies using an established NCI-N87 colon cancer model (therapeutic setting), a single injection of the Araris Topo 1 ADC at DAR2 at a dose of 52ug/kg (adjusted payload dose) induced superior anti-tumor activity compared to Trastuzumab deruxtecan at DAR of 8, injected at the same payload dose. Complete tumor regression of all tumors (7/7) was obtained at 104ug/kg payload dose and lasted throughout the whole study duration (total 80 days) and was very well tolerated. The data show that Araris Topo 1 ADCs assembled using novel peptide linkers, even at a DAR of as low as 2 have a very efficient anti-tumor activity suggesting optimal drug exposure, targeting and release of the payload. In summary, we show that the Araris Topo1 linker-payloads result in highly potent ADCs with very favorable biophysical properties and extremely efficient payload release as well as an antibody-like exposure profile making them ideal linker-payloads for solid tumor targeting. We anticipate the low-drug load to be favorable in avoiding excessive toxicities in non-targeted tissues. Finally, the Araris bioconjugation technology allows for the generation of tailor-made ADC candidates with improved therapeutic indices.

Abstract LB221: Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses

The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia.

Abstract LB227: Leveraging novel Dato-DXd resistance models to inform biomarker discovery and rational combinations to combat drug resistance

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 (TOP1) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Despite its promising early clinical signals, drug resistance is a challenge that may emerge with time. The mechanisms of Dato-DXd resistance are currently unknown. Herein, we created and investigated novel models of Dato-DXd resistance to characterize resistance mechanisms and identify novel combinations to overcome Dato-DXd resistance. We used a cyclical dosing method to develop two Dato-DXd acquired resistant NCI-N87 gastric cancer cell line models from in vitro cells (N87-IVR) and in vivo tumors (N87-EVR). The N87-IVR and N87-EVR cells were found to be more than 40-fold resistant to Dato-DXd compared to the parental N87 cells. First, we assessed TROP2 levels, Dato-DXd binding and expression of drug efflux pumps known to have TOP1-inhibitor substrates between the parental and resistant cell populations. Minor changes in each of these were not sufficient to drive Dato-DXd resistance in the drug-tolerant models. Subsequently, we performed a proteogenomic analysis to identify which proteins and pathways were modulated in the resistant phenotype. Both the N87-IVR and N87-EVR cells were divergent from the parental N87 cells and, interestingly, they showed similar yet distinct profiles. Among other changes, our analysis revealed a dramatic loss of SLFN11, a putative DNA/RNA helicase, in both resistant models. Previously published data has shown that a combination with an ATR inhibitor (ATRi) can re-sensitize TOP1 inhibitor-resistant cells in SLFN11-low settings. Consistent with these data, we observed that the ATRi ceralasertib (AZD6738) in combination with Dato-DXd re-sensitizes the resistant NCI-N87 cells in vitro and in vivo. In addition to SLFN11 loss, we also identified a number of other modulations with the potential to drive resistance, e.g. downregulation of the caveolin internalization machinery and interferon response genes. These findings help us refine the mechanism of action of Dato-DXd and shed light on the potential mechanisms of Dato-DXd resistance that may emerge clinically. These data have been used to uncover new biomarkers of Dato-DXd sensitivity and can lead to the development of new clinical strategies to combat Dato-DXd resistance, e.g. the combination with ATRi.

Abstract LB245: ONM-501, a dual-activating polyvalent STING agonist, enhances tumor retention and demonstrates favorable preclinical safety profile

Background: The Stimulator of Interferon Genes (STING) plays a crucial role in the innate immune response. Several previous STING agonist development compounds have shown limited therapeutic efficacy in oncology clinical trials. ONM-501 is a novel STING agonist: the endogenous STING agonist 2’,3’-cyclic GMP-AMP (cGAMP) is encapsulated within PC7A micelles. PC7A induces polyvalent STING condensation and prolongs immune activation. cGAMP-PC7A nanoparticles offer a dual ‘burst’ and ‘sustained’ STING activation. The anti-tumor efficacy and pharmacodynamic analysis of ONM-501 in multiple tumor models have been demonstrated previously. Here we report the pharmacokinetic (PK) and biodistribution (BD) analysis of ONM-501 in mice and safety evaluation in mice, rats and primates.

Methods: PC7A polymers conjugated with LiCOR 800CW were mixed with unlabeled PC7A in 1:9 ratio and cGAMP was encapsulated into micelles to generate an “always-on” fluorescently labelled ONM-501-CW800. Naïve or tumor-bearing mice were injected subcutaneously (SC) or intratumorally (IT) with ONM-501-CW800, respectively, and plasma and multiple organ samples were collected; the whole tissue specimens were first imaged ex vivo using LiCOR Pearl Imaging system, and then homogenized and the fluorescence quantified against standard curves prepared by spiking ONM-501-CW800 into a homogenate of the relevant matrix. PK parameters were calculated using non-compartmental methods. Safety and tolerability were evaluated by single- and multiple-dose SC injections in naïve animals up to the highest feasible doses.

Results: The BD pattern of ONM-501-CW800 was similar after IT and SC injections. The highest concentrations were observed at the injection sites and draining lymph nodes at all timepoints for both routes of administration. The concentrations in the injection site were much higher in tumors following IT than in dermal tissue following SC injection. After a 50 µg dose, systemic exposure to ONM-501-CW800 was ~1.8- and 2.4-fold lower after IT than SC injection based on Cmax and AUC(inf), respectively. The plasma t½ after IT injection, 17.4 hours, was ~1.3-fold longer than after SC injection, 12.9 hours. The Cmax and AUC (inf) in tumors were ~144- and 120-fold higher than in plasma after IT injection, with a t½ of 25.2 hours in tumors. In single-dose toxicology studies, ONM-501 was well tolerated in mice, rats and monkeys without severe or irreversible systemic toxicities up to the maximum feasible SC doses at 74, 45 and 30 mg/kg, respectively. In the 4-week repeat-dose GLP toxicology studies, the highest non-severely toxic SC dose (HNSTD) was 30 and 7.5 mg/kg in rats and monkeys, respectively.

Conclusions: Systemic exposure to ONM-501 was lower after IT than SC administration, which is consistent with increased ONM-501 retention in tumors. Combined with preclinical toxicology studies, ONM-501 showed a favorable pharmacokinetic, tolerability and safety profile that support its continued development in cancer patients.

Abstract LB246: Enfortumab vedotin, a nectin-4-directed antibody-drug conjugate, demonstrates compelling antitumor activity in non-muscle invasive bladder cancer models which predicts minimal systemic exposure when administered by intravesical instillation in patients

Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate directed to Nectin-4, which is highly expressed in bladder cancers. Preclinically, EV has demonstrated tumor cell killing by direct cytotoxicity and bystander effect and can induce the hallmarks of immunogenic cell death. EV improves survival in adults with previously treated locally advanced or metastatic urothelial carcinoma (la/mUC) and is approved in the US, Europe, Japan, and others. Most newly diagnosed bladder cancer cases are non-muscle invasive (NMIBC). Standard treatment of high-risk NMIBC involves transurethral resection followed by intravesical Bacillus Calmette-Guerin (BCG) or chemotherapy. Although response to BCG is high, recurrence is common, and treatment options for patients with BCG-unresponsive tumors are limited, underscoring the significant unmet need. Previously, we demonstrated compelling preclinical antitumor activity of EV in NMIBC models with a favorable safety profile and minimal systemic exposure. EV-mediated antitumor activity was confirmed in a mouse model of NMIBC by both bioluminescence imaging and IHC for hNectin-4-expressing cancer cells. Following intravesical administration of EV, tumor growth inhibition ranged 46-96% across the dose range tested. Colocalization of EV to Nectin-4-positive tumor tissues was confirmed by IHC in the engrafted tumor cells. Systemic EV exposure in tumor-bearing mice was low, consistent with previous nonclinical studies, supporting that the antitumor activity is driven by local exposure within the bladder. In a repeat-dose GLP toxicology study in rats, no systemic toxicities were observed at intravesical doses up to 6-fold higher than the maximum tolerated IV dose. This lack of systemic toxicities that can occur with IV administration in rats was likely due to minimal systemic exposure of both EV and unconjugated MMAE. Currently, the safety, tolerability, and antitumor activity of intravesical EV are being evaluated in a Phase 1 study in adults with high-risk, BCG-unresponsive NMIBC (EV-104, NCT05014139). The initial dose level for EV-104 was selected to be active and predicted to have minimal systemic absorption based on preclinical and known clinical IV data. Here, we present confirmatory clinical data demonstrating that EV and unconjugated MMAE are undetectable in the bloodstream at the starting dose. These findings confirm the translatability of our nonclinical models and provide evidence that intravesical administration of EV in NMIBC is a promising approach that limits systemic exposure. These data support the potential for a favorable safety and activity profile and warrant continued investigation of intravesical EV in patients with NMIBC.

声明：以上内容仅供参考，不构成投资建议。

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