Control Antibody-drug Conjugate Research Articles

Abstract 1911: Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model

Abstract Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 monoclonal antibody linked to a potent DNA topoisomerase I (TOP1) inhibitor payload via a plasma-stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd is currently under clinical investigation for the treatment of patients with solid tumors including non-small cell lung cancer (NSCLC), hormone receptor positive (HR+ BC) and triple negative breast cancer (TNBC). Pharmacotherapy of brain tumors can be limiting due to restricted drug delivery across blood brain and blood tumor barrier. Enhertu®, that uses the same DXd ADC technology, has reported clinical activity in patients with brain metastases from HER2+ breast cancer, but very little information is available on what drives ADC biodistribution and activity in CNS-involved cancers. Here, we investigated whether systemically administered ADCs can penetrate the brain microenvironment and mediate anti-tumor activity in a preclinical model. Luciferase-tagged H1373 (TROP2-expressing NSCLC) tumor cells were intracranially implanted into NSG mice. Tumor-bearing mice were dosed with Dato-DXd or matched isotype Control IgG-ADC (DAR4) at 10mpk 7 days or 14 days post intracranial tumor implant. Dato-DXd inhibited intracranial tumor growth better than Control ADC (Day 7: 105% TGI vs 38% TGI; Day 14: 65% TGI vs <10% TGI, respectively, compared to Vehicle). Immunohistochemistry analysis of brain tissue validated localization of Dato-DXd in the tumor, suggesting Dato-DXd can distribute into the local tumor microenvironment in this preclinical tumor model. Additionally, treatment with Dato-DXd provided a significant survival benefit over Control ADC (Median survival of 63 days vs 43 days, P=0.0002). This preclinical study supports the inclusion of Dato-DXd in treatment of patients with CNS-involved tumors. Understanding the pharmacological determinants of Dato-DXd activity in the CNS will help outline strategies to implement Dato-DXd-based treatment of patients with CNS-involved tumors. Citation Format: Kristen Jones Jones, Montira Suksomboon, SaraAnn rosenthal, Jacob Gordon, Corinne Reimer, Matthew Sung, Chetan Rane. Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1911.

Abstract 6341: Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE

Abstract Background: Myeloid epithelial reproductive tyrosine kinase (MERTK), a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family, is overexpressed in cancer and associated with reduced apoptosis and chemoresistance, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) (Inspirna, Inc), selectively binds to MERTK with high affinity on tumor cells, resulting in internalization and degradation of the receptor. It then induces cytotoxicity through the release of the payload, MMAE (monomethyl auristatin E), which disrupts mitosis. We hypothesize that targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: First, to determine the clinical relevance of MERTK, using the Oregon Health and Science University (OHSU) acute myeloid leukemia (AML) dataset, we divided patients into low and high MERTK mRNA expression based on mean expression and compared their overall survival. To identify the AML subtypes with the highest MERTK expression, we analyzed samples from 810 AML patients at MD Anderson using reverse phase protein arrays (RPPA), as well as 8 AML cell lines and the peripheral blood of 9 AML patients using flow cytometry. Then, the cell lines with the highest MERTK expression were treated with varying doses of RGX-019-MMAE or isotype control for 120 hours and assessed for viability with CellTiter-Glo 2.0. Likewise, we administered RGX-019-MMAE to primary cells from AML patients and determined the anti-leukemic effect. Finally, we investigated the synergistic effect of RGX-019-MMAE with venetoclax, a BCL2 inhibitor, or cytarabine, a chemotherapy agent, in vitro. Results: In the AML OHSU dataset, patients with high MERTK mRNA expression had significantly worse overall survival (p=0.02). RPPA revealed that patients with PTPN11 mutation, t (9;11), and monocytic AML subtypes had significantly higher MERTK protein expression. MERTK protein expression varied in AML cell lines and was highest in Kasumi-1 and OCI-AML3. Treatment of these two cell lines with RGX-019-MMAE resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p<0.01). Similarly, in primary AML cells with high MERTK expression, RGX-019-MMAE treatment induced significant cell death compared to the control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated with RGX-019-MMAE combined with venetoclax or cytarabine showed a synergistic cytotoxic effect in a dose-dependent manner. Conclusion: High MERTK expression in monocytic AML suggested that MERTK is a promising therapeutic target in this subtype. RGX-019-MMAE significantly induced AML cell death and produced a synergistic anti-leukemic effect in combination with chemotherapeutic agents and targeted therapy in vitro, suggesting its potential for targeting patients with high-risk AML. Citation Format: Anudishi Tyagi, Maryam Siddiqui, Isabel Kurth, Shugaku Takeda, Amanda Eckstrom, Jenny Borgman, Abhishek Maiti, Venkata Lokesh Battula. Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6341.

Abstract 4551: A novel MET antibody-drug conjugate based combination therapy to overcome colorectal cancer plasticity and drug resistance

Abstract Despite therapeutic advancements, colorectal cancer (CRC) remains the second deadliest malignancy in the United States. Challenges encountered in CRC treatment may be attributed to cancer stem cells (CSC), an immortal tumor cell population. CSCs potentiate tumor relapse by exploiting its infinite replicative potential and inherent drug resistance. Moreover, CSCs exhibit plasticity, allowing them to transition between differentiated and undifferentiated states in response to environmental cues to evade therapy and drive metastatic progression. MET is a receptor tyrosine kinase frequently upregulated in CRCs. We previously demonstrated that treatment with chemotherapies or antibody-drug conjugates (ADCs) targeting the CSC marker LGR5 led to the loss of LGR5 expression with concomitant MET-STAT3 pathway activation in therapy-resistant CRC cells. Further, we showed that LGR5 couples to the scaffold protein IQGAP1, which correlates with poor prognosis in several cancer types. Interestingly, our new data revealed that LGR5 knockdown enhances IQGAP1 interaction with MET and STAT3 via co-immunoprecipitation assays, suggesting a role for IQGAP1 in mediating MET-STAT3 activation and plasticity of LGR5+ CRC cells. To overcome therapy-induced plasticity, we generated MET-targeting ADCs conjugated to DNA-crosslinking payloads. First, we cloned and produced MET monoclonal antibodies (mAbs) and evaluated them for specificity and binding in a panel of CRC cell lines expressing different MET protein levels. MET mAbs demonstrated high selectivity for MET-expressing CRC cells and internalized to lysosomes, which is necessary for ADC payload release. To generate MET ADCs, we employed a site-specific conjugation methodology to attach cleavable peptide linker-drug moieties to the highest affinity MET mAb. MET ADCs were evaluated for cancer cell-killing efficacy in vitro in parallel with MET mAb, non-targeting control mAb (cmAb), and control ADC (cADC). Our MET ADC displayed high potency and efficacy in MET-expressing CRC cells, whereas MET mAb, cmAb, and cADC had minimal effects. MET ADCs were then tested in combination with chemotherapies or other targeted therapies. The combination of MET ADCs with 5-fluorouracil showed a synergistic effect in vitro. Future work involves investigating the safety and efficacy of MET ADCs alone and combined with chemotherapies in xenograft models of CRC. These findings present a mechanism underpinning CRC plasticity and the rationale for a novel treatment modality to potentially overcome CRC resistance and relapse. Citation Format: Shraddha Subramanian, Tressie A. Posey, Joan Jacob, Adela Aldana, Kendra S. Carmon. A novel MET antibody-drug conjugate based combination therapy to overcome colorectal cancer plasticity and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4551.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6579.

Abstract 3122: Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6

Abstract Claudin-6 is a member of the claudin family and consists of four transmembrane helices, two extracellular loops, and an amino- and carboxyl-terminal tail with a PDZ-binding motif in the cytoplasm. It is involved in the formation of tight junctions and is expressed in developing human epithelial structures during early-to-mid gestation while expression in iadult tissues is mostly absent. Claudin-6 expression is upregulated in ovarian cancer, testicular cancer, endometrial cancer, non-small cell lung cancer (NSCLC) and gastric cancer. Here, the generation and preclinical characterization of a novel Claudin-6 specific antibody-drug conjugate (ADC) is described, composed of a humanized IgG1 directed against human Claudin-6 site-specifically conjugated a novel payload containing a valine-alanine cleavable linker and a camptothecin warhead with a drug to antibody ratio of 6. FcγR-mediated effector function of the ADC was abrogated via mutations in the Fc portion of the antibody. The purpose of this study was to evaluate the expression of Claudin-6 in different tumour indications and characterise the in vitro and in vivo anti-tumour activity in human cancer cell lines and patient-derived xenograft (PDX) models. In addition, the tolerability of the ADC was evaluated in a repeat-dose toxicity study cynomolgus monkeys. Cell binding studies using Fabs showed high affinity binding to Claudin-6, while affinity for Claudin-9 was 16-fold lower with no binding to Claudin-3 and Claudin-4. Cross-reactivity with cynomolgus monkey and mouse Claudin-6 was also demonstrated. Fcgamma receptor binding studies confirmed the strongly reduced binding of the antibody to FcγI, FcγI,IIa, FcγIIb and FcγIIa, consistent with the introduction of mutations in the Fc domain of the antibody. In vitro, the Claudin-6 ADC showed potent and specific cytotoxicity against various Claudin-6-expressing solid cancer cell lines, including OVCAR3, PA-1, OV-90, FU-97, BeWo, NTERA-2 and NUGC3 in comparison to a non-binding control ADC conjugated to the same payload, and it also showed potent bystander killing. In vivo, a single dose intravenous (IV) of the Claudin-6 ADC demonstrated potent anti-tumour activity in multiple solid tumour xenograft models, including OVCAR3 and PA-1. Furthermore, administration of the Claudin-6 ADC to cynomolgus monkeys was well tolerated up to 40 mg/kg IV when given on Day 1 and 22. Cell membrane expression of Claudin-6 was confirmed by immunohistochemistry (IHC) in ovarian cancer (70%), testicular cancer (98%), endometrial cancer (30%), NSCLC (28%) and gastric cancer (24%), confirming expression of Claudin-6 in indications with high unmet need. In summary, this novel Claudin-6 ADC demonstrated potent, specific anti-tumour activity in Claudin-6 expressing cancer-derived models and was well tolerated in cynomolgus monkey, supporting future clinical development of this ADC. Citation Format: Nicola Tsang, Nicolas Veillard, Elizabeth Horsley, Karin Havenith, Narinder Janghra, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Paul W. Hogg, Francesca Zammarchi, Lolke de Haan, Patrick H. van Berkel. Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3122.

IKS03, a Novel CD19-Targeted Antibody Drug Conjugate, Induces Target Dependent In Vivo Cell Killing of B-Cell Lymphoma Xenografts By DNA Crosslinking

CD19 is widely expressed in B-cell non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). IKS03 is a CD19-targeting antibody drug conjugate (ADC) with a novel linker payload design for tumor-selective payload release. Site-specific conjugation using farnesyltransferase-catalysed ConjuALL technology to a DNA crosslinking agent yields a homogenous ADC with drug-to-antibody ratio (DAR) of 2. To minimize systemic release of the highly potent payload in human plasma, IKS03 incorporates a linker specifically cleavable by the lysosomal enzyme beta-glucuronidase. In addition, the payload is a pro-drug with a protecting moiety cleavable by the same enzyme. Therefore, following CD19-dependent binding and uptake, IKS03 requires intracellular lysosomal processing in the target cell to both release and fully activate the free pyrrolobenzodiazepine (PBD) dimer payload which induces DNA crosslinking, blocks DNA replication and ultimately leads to tumor selective cell death. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. IKS03 was highly active against Farage and OCI-LY10 DLBCL xenografts as well as Granta-519 MCL xenografts in SCID mice with durable tumor regressions observed following treatment with a single dose of 0.1 to 0.3 mg/kg. Likewise, in a Ramos lymphoma model a single IKS03 dose of 0.3 mg/kg significantly inhibited tumor growth. IKS03 demonstrated greater in vivo efficacy than the clinical benchmark ADCs polatuzumab vedotin and loncastuximab tesirine approved for the treatment of patients with B-cell non-Hodgkin lymphomas. PBD cytotoxic agents have been described to induce DNA crosslinking leading to DNA damage. To demonstrate that IKS03 in vivo activity is the result of targeted payload release, Ramos xenografts were established in immunodeficient mice and treated with up to 1 mg/kg of IKS03 in comparison with a non-targeted ADC utilizing the same linker payload format. Tumors were excised at 1 day and 3 days post-treatment and assessed for the presence of phospho-H2AX positive foci, a marker of DNA damage, by immunohistochemistry. IKS03 treatment increased both frequency and staining intensity of nuclear phospho-H2AX foci in Ramos xenografts in a dose- and time-dependent manner. In contrast, the non-targeted control ADC did not result in any changes as compared to a vehicle control. This confirms the specific induction of DNA damage in CD19 positive lymphomas as a pharmacodynamic response to IKS03 treatment in vivo. In conclusion, IKS03 is highly effective in an array of preclinical MCL and DLBCL xenograft models at doses that are well tolerated. In vivo, IKS03 treatment increased the level of phospho-H2AX indicative of irreversible DNA damage in a target-dependent manner. This is consistent with the proposed mechanism of action of CD19-mediated delivery of a PBD payload specifically to B-cell tumors. Clinical investigation in patients with advanced B-Cell Non-Hodgkin Lymphomas is being initiated (NCT05365659).

Targeting Myeloid Epithelial Tyrosine Kinase (MERTK) Receptor in Acute Myeloid Leukemia Using a Novel Antibody Drug Conjugate, Rgx-019-MMAE

Background: Myeloid epithelial reproductive tyrosine kinase receptor (MERTK) is a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family. It is known to be overexpressed in various solid and hematological cancers. Overexpression of MERTK is associated with reduced apoptosis, increased metastasis, and drug resistance in tumor cells, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) developed by Inspirna, selectively binds to MERTK on tumor cells, which results in internalization and degradation of the receptor, and induces cytotoxicity through the release of the payload, MMAE, which disrupts the mitosis process of actively dividing cells. Several monoclonal antibodies targeting MERTK have shown promising results against various solid cancers, however, the effect of targeting MERTK in AML progression via RGX-019-MMAE is not known. Here, we hypothesize MERTK is overexpressed in distinct subsets of AML and associated with leukemia progression, therefore, targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: To determine the clinical/prognostic relevance of MERTK, we analyzed a gene expression dataset (OHSU) and segregated the patients into low and high MERTK mRNA expression groups based on their mean expression value. To identify the specific AML subset with the highest MERTK expression, MERTK protein expression was analyzed in 818 patients with AML by performing Reverse Phase Protein Arrays (RPPA). MERTK protein expression was also measured by flow cytometry in OCI-AML3, OCI-AML2, Kasumi-1, ThP-1, Molm-13, Molm-14, MV4-11, and U937 leukemic cell lines and in the peripheral blood or bone marrow of five AML patients. Further, we treated Kasumi-1 and OCI-AML3 AML cell lines with varying doses of RGX-019-MMAE or isotype control for 72 and 120 hours and measured its effect on AML cell viability using CellTiterGlo 2.0 assay. Likewise, we treated primary cells from 5 different AML patients and determined the anti-leukemic effect of RGX-019-MMAE in primary cells. Finally, we investigated the synergistic effect of RGX-019-MMAE with Venetoclax (BCL2 inhibitor) or 5-Azacytidine in vitro. Results: Analysis of AML OHSU dataset showed that patients with high MERTK mRNA expression had significantly worse overall survival than those having low MERTK mRNA expression (p=0.02). RPPA analysis revealed significantly higher MERTK protein expression in monocytic AML subtypes, such as M4 and M5, especially in those with PTPN11 mutation or t (9;11) translocation. Moreover, in the RPPA we noted that high expression of MERTK protein levels is significantly correlated with inferior survival. This finding suggests that MERTK could serve as a therapeutic target in AML, especially in patients with monocytic subtypes of leukemia. In addition, we observed varying degrees of MERTK protein expression in AML cell lines, with Kasumi-1 and OCI-AML3 expressing the highest amount of MERTK protein. Next, treatment of Kasumi-1 and OCI-AML3 cell lines with varying doses of anti-MERTK ADC, RGX-019-MMAE, resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p<0.01), suggesting that RGX-019-MMAE inhibits AML cell proliferation. Further, we validated this finding in primary AML cells with high MERTK expression. As expected, we observed that treatment with RGX-019-MMAE induced significantly greater cell death (˜80% apoptosis) in leukemic cells than the isotype control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated in combination with RGX-019-MMAE and either Venetoclax or 5-Azacytidine showed a synergistic effect with increased cytotoxicity in a dose-dependent manner. These findings suggest that RGX-019-MMAE targets MERTK-expressing leukemic cells, sensitizes cells to chemotherapies, and promotes overall cytotoxicity. Conclusion: RGX-019-MMAE significantly induced cell death in AML cell lines and primary AML patient samples. RGX-019-MMAE, combined with chemotherapeutic agents and targeted therapy, produces a synergistic anti-leukemic effect. Our data indicates that MERTK is a potential therapeutic target in AML patients with monocytic subtypes of leukemia.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

Preclinical radiolabeling, in vivo biodistribution and positron emission tomography of a novel pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2

IntroductionAnti-ASCT2 antibody drug conjugate (ADC) MEDI7247 has been under development as a potential anti-cancer therapy for patients with selected relapsed/refractory hematological malignancies and advanced solid tumors by MedImmune. Although promising efficacy was observed in the clinic, pharmacokinetic (PK) analyses observed low exposure of MEDI7247 in phase I hematological patients. To investigate the biodistribution properties of MEDI7247, MEDI7247 and control antibodies were radiolabeled with zirconium-89 and in vitro and in vivo properties characterized. MethodsMEDI7247 (human anti-ASCT2 antibody conjugated with pyrrolobenzodiazepine (PBD)) and MEDI7519 (MEDI7247 without PBD drug conjugate) and an isotype control antibody drug conjugate construct were conjugated with p-isothiocyanatobenzyl-deferoxamine (Df) and radiolabeled with zirconium-89. In vitro studies included determining the radiochemical purity, protein integrity, immunoreactivity (Lindmo analysis), apparent antigen binding affinity for ASCT2-positive cells by Scatchard analysis and serum stability of the radiolabeled immunoconjugates. In vivo studies included biodistribution and PET/MRI imaging studies of the radiolabeled immunoconjugates in an ASCT2-positive tumor model, HT-29 colorectal carcinoma xenografts. ResultsConditions for the Df-conjugation and radiolabeling of antibody constructs were determined to produce active radioimmunoconjugates. In vivo biodistribution and whole body PET/MRI imaging studies of [89Zr]Zr-Df-MEDI7519 and [89Zr]Zr-Df-MEDI7247 radioimmunoconjugates in HT-29 colon carcinoma xenografts in BALB/c nude mice demonstrated specific tumor localization. However, more rapid blood clearance and non-specific localization in liver was observed for [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 compared to isotype control ADC. Except for liver and bone, other normal tissues demonstrated clearance reflecting the blood clearance for all three constructs and no other abnormal tissue uptake. Conclusions and advances in knowledgePreclinical biodistribution analyses of [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 showed the biodistribution pattern of anti-ASCT2 ADC MEDI7247 was similar to parental MEDI7519, and both antibodies showed specific tumor uptake compared to an isotype control ADC. This study highlights an important role nuclear medicine imaging techniques can play in early preclinical assessment of drug specificity as part of the drug development pipeline.

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

Abstract 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. Citation Format: Xuemei Xie, Jangsoon Lee, Jon A. Fuson, Huey Liu, Young J. Gi, Thanasis Poullikkas, Pang-Dian Fan, Kumiko Koyama, Debu Tripathy, Naoto T. Ueno. Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd)in tamoxifen-resistant ER+ breast cancer cells by inducing DNA damage and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB042.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

284 (PB064) - Ultrasound-induced cavitation enhances therapeutic efficacy of AXL-targeting ADC leading to improved survival in a human xenograft model of renal cancer

Background: Antibody-drug conjugates (ADCs) are a new class of antitumor agents that combine the targeting specificity of monoclonal antibodies (mAbs) with the cell killing ability of powerful cytotoxic molecules. However, poor penetration of drugs into solid tumors, including mAbs, is a well-recognized barrier to effective therapy as this strongly limits the number of tumor cells targeted by the therapeutic agent. A novel non-invasive approach to tackle this problem is ultrasound-mediated drug delivery using co-infused cavitation nuclei, which shows promise as a way to pump more drug (including mAbs/ADCs) into and throughout tumors. The purpose of this study was to test a suboptimal single dose of mipasetamab uzoptirine (ADCT-601), an ADC targeting the AXL protein, in combination with ultrasound-induced cavitation in a preclinical mouse model of renal cancer. Material and methods: SCID mice were injected subcutaneously with SN12C human renal cancer cells and when tumors reached an average size of 150 mm3, animals were divided into 6 cohorts (N = 6–7 mice each): a) PBS (vehicle control); b-c) B12-PL1601 (control ADC) at 0.3 mg/kg ± cavitation; d–e) ADCT-601 at 0.3 mg/kg ± cavitation; f) ADCT-601 at 1 mg/kg (efficacious dose). Cavitation nuclei and drug agent were administered intravenously just before ultrasound application from the OxSonics SonoCart system (ultrasound parameters: fc = 0.5 MHz; 8000cycles; PRF = 0.5 Hz; PRFP = 1–2.9 MPa variable). Ultrasound amplitude was set according to the cavitation monitoring method (passive acoustic mapping [PAM]) to be within 0.1–0.5 nJ/pulse for 10 minutes of treatment. Mice were culled when tumors’ width + length passed the value of 24 mm. Results: Results indicated that cavitation was achieved in the desired range for all ultrasound groups. Following treatment, 2 groups (ADCT-601 at 1 mg/kg and ADCT-601 at 0.3 mg/kg + cavitation) showed significant tumor growth delay, ultimately leading to a significant improvement in survival when compared to all other groups. No significant difference between the high and the low ADCT-601 dose with cavitation was observed, indicating that the combination enhanced potency by 3.3-fold. Conclusions: Cavitation-enhanced delivery of ADCT-601 has a strong effect on xenograft tumor growth leading to a significant increase in survival. This was demonstrated for a suboptimal ADC dose, indicating that ultrasound-mediated drug delivery could be used to reduce the administered dose while preserving therapeutic efficacy. These preclinical data warrant further evaluation of this promising combination in the clinic. Conflict of interest: Ownership: Massimo Masiero, Erika Vojtasova, Paul Boulos, Calum Crake, Christian Coviello and Cliff Rowe own OxSonics Therapeutics share options. Christian Coviello also owns OxSonics Therapeutics shares. Francesca Zammarchi and Patrick van Berkel own ADC Therapeutics shares. Other Substantive Relationships: Massimo Masiero, Erika Vojtasova, Paul Boulos, Calum Crake, Christian Coviello and Cliff Rowe are OxSonics Therapeutics employees. Christian Coviello is also OxSonics Therapeutics co-founder. Francesca Zammarchi and Patrick van Berkel are ADC Therapeutics employees.

In Vivo and In Vitro Efficacy of Enhertu in Uterine Serous Carcinoma (047)

Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Graphical Abstract View Large Image Figure Viewer Download Hi-res image Graphical Abstract View Large Image Figure Viewer Download Hi-res image Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted. Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted.

Trastuzumab deruxtecan (DS-8201a), a HER2-Targeting Antibody-Drug Conjugate with Topoisomerase I Inhibitor Payload, Shows Antitumor Activity in Uterine and Ovarian Carcinosarcoma with HER2/neu Expression (046)

<b>Objectives:</b> Carcinosarcomas (CSs) are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression. We evaluated the preclinical <i>in vitro</i> and <i>in vivo</i> activity of trastuzumab deruxtecan (DS-8201a), a novel HER2/neu-targeting antibody-drug conjugate (ADC) against primary uterine and ovarian CSs overexpressing HER2/neu<i>.</i> <b>Methods:</b> Twelve primary uterine and ovarian CS cell lines were evaluated for HER2/neu surface expression by immunohistochemistry (IHC) and by flow cytometry, and gene amplification by ¹³cytotoxicity, viability, and bystander killing effect assays. <i>In vivo</i> activity was studied in mouse CS xenograft models. <b>Results:</b> DS-8201a, as compared to MAAA-9199 control ADC, was found to be highly effective against uterine and ovarian CS cell lines demonstrating 3+ HER2/neu expression. Specifically, the mean half-maximal inhibitory concentrations (IC50's) were 0.02931 μg/mL and 9.75 μg/mL (p<0.0001) against 3+ HER2/neu uterine cell line, and 0.0285 μg/mL and 20.9 μg/mL (p<0.0001) against 3+ ovarian HER2/neu cell lines for DS-8201a versus MAAA-9199, respectively. There was no significant difference between DS-8201a and MAAA-9199 against CS cell lines with low/negligible HER2/neu expression. Importantly, DS-8201a induced an efficient bystander killing effect of HER2/neu negative tumor cells when admixed with 3+ HER2/neu cells. <i>In vivo</i> studies confirmed that DS-8201a was significantly more effective than MAAA-9199 in decreasing tumor volume and prolonging life in 3+ HER2/neu-expressing CS xenografts (attached Figure). There was no significant toxicity demonstrated between treatment and control groups. <b>Conclusions:</b> Trastuzumab deruxtecan (DS-8201a) is a novel ADC with remarkable activity against CS with strong (3+) HER2/neu expression. Clinical studies with DS-8201a in patients harboring chemotherapy-resistant CS with high HER2/neu expression are warranted. DS-8201a showing significant tumor growth inhibition in 3+ HER2/neu expression mice. Day 8 mean tumor volumes in treated mice with DS-8201a (0.271 cm3) versus ADC isotope control (0.859 cm3, p=0.0011) and vehicle control (0.804 cm3, p=0.0047). Day 11 mean tumor volumes in treated mice with DS-8201a (0.179 cm3) versus ADC isotope control (1.166 cm3, p <0.0001) and vehicle control (1.151 cm3, p <0.0001).Fig. 1

Design and Characterization of Immune-Stimulating Imidazo[4,5-c]quinoline Antibody-Drug Conjugates.

Traditional antibody-drug conjugate (ADC) technology has employed tumor-targeting antibodies to selectively deliver ultrapotent cytotoxins to tumor tissue. While this technology has been highly successful, resulting in the FDA approval of over 10 ADCs, the field continues to struggle with modest efficacy and significant off-target toxicity. Concurrent with the struggles of the ADC field, a new generation of immune-activating therapeutics has arisen, most clearly exemplified by the PD-1/PD-L1 inhibitors that are now part of standard-of-care treatment regimens for a variety of cancers. The success of these immuno-oncology therapeutic agents has prompted the investigation of a variety of new immuno-stimulant approaches, including toll-like receptor (TLR) activators. Herein, we describe the optimization of ADC technology for the selective delivery of a potent series of TLR7 agonists. A series of imidazole[4,5-c]quinoline agonists (as exemplified by compound 1) were shown to selectively agonize the human and mouse TLR7 receptor at low nanomolar concentrations, resulting in the release of IFNα from human peripheral blood mononuclear cells (hPBMCs) and the upregulation of CD86 on antigen-presenting cells. Compound 1 was attached to a deglycosylated (Fc-γ null) HER2-targeting antibody via a cleavable linker, resulting in an ADC (anti-HER2_vc-1) that potently and selectively activated the TLR7 pathway in tumor-associated macrophages via a "bystander" mechanism. We demonstrated that this ADC rapidly released the TLR7 agonist into the media when incubated with HER2+ cells. This release was not observed upon incubation with an isotype control ADC and furthermore was suppressed by co-administration of the naked antibody. In co-culture experiments with HER2+ HCC1954 cells, this ADC induced the activation of the NFκB pathway in mouse macrophages and the release of IFNα from hPBMCs, while a corresponding isotype control ADC did not. Finally, we demonstrated that IP administration of anti-HER2_vc-1 induced complete tumor regression in an HCC1954 xenograft study in SCID beige mice. Unlike related ADC technology that has been reported recently, our technology relies on the passive diffusion of the TLR7 agonist into tumor-associated macrophages rather than Fc-γ-mediated uptake. Based on these observations, we believe that this ADC technology holds significant potential for both oncology and infectious disease applications.

Abstract 4251: Development and characterization of an epiregulin antibody-drug conjugate for targeting colorectal cancer cell plasticity

Abstract Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. This is largely due to metastasis, tumor relapse, and the vast heterogeneity of CRC tumors leading to treatment failure. Cancer stem cells (CSCs) are a small population of tumor-initiating cells thought to act as immortal seeds that can form metastases and contribute to tumor relapse. CSCs have been shown to exhibit plasticity or the ability to transition between differentiated and undifferentiated states to evade treatment and to promote tumor progression. Epiregulin (EREG) is an EGFR family ligand that is highly expressed in both states, as well as in treatment resistant CRCs of various mutation statuses. We showed that knock-out of EREG in a CRC cell line resulted in significant tumor inhibition in vivo, suggesting EREG is a promising target for therapeutic development. Therefore, we generated EREG-targeted antibody-drug conjugates (ADCs) to act as guided missiles for the delivery of cytotoxic drugs into EREG-expressing tumors to overcome plasticity and resistance in CRC. To develop a novel EREG ADC, we cloned and produced three separate EREG monoclonal antibody (mAbs) and tested them for binding, the ability to internalize into CRC cells, and to neutralize EREG activity. All three EREG mAbs were shown to bind with high affinity to EREG tethered to the surface of CRC cells and internalize to the lysosome, which is important for processing the release of drug from the ADC. We then conjugated the highest affinity EREG mAb and a non-targeting isotype control antibody to a highly cytotoxic drug via a cleavable peptide linker. EREG ADCs were evaluated in vitro against a wide panel of CRC cell lines of various Kras, Braf, and PI3CKA mutational statuses and different levels of EREG expression. Drug efficacy was determined using cytotoxicity assays to measure changes in CRC cell survival with escalating doses of unconjugated control antibody and EREG mAb, as well as control ADC (cADC) and EREG ADC. EREG mAb alone did not result in significant CRC cell death. However, EREG ADCs exhibited potent cell-killing in EREG-expressing CRC cells, whereas minimal effects were observed with cADC. Future progress will involve testing the safety and efficacy of EREG ADCs against patient-derived tumor xenograft models of CRC in vivo. These preliminary results demonstrate the therapeutic potential of EREG ADCs for targeting plasticity and drug resistance in CRC. Citation Format: Joan Jacob, Zhengdong Liang, Kendra Carmon. Development and characterization of an epiregulin antibody-drug conjugate for targeting colorectal cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4251.

Abstract LB088: Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression

Abstract Background: HER3, a member of the ErbB family of receptor tyrosine kinases that activate multiple oncogenic signaling pathways, is overexpressed in 50-70% of breast cancers (BC). HER3 mRNA levels are higher in ER+ breast tumors than in other molecular subtypes. Inhibition of ER activity using an antagonist increased HER3 protein expression and activation, which was essential for growth and survival of ER+ BC cells resistant to the ER antagonist. Therefore, therapeutically targeting HER3 with HER3-DXd, an antibody-drug conjugate (ADC) composed of a fully human anti-HER3 monoclonal antibody (patritumab) conjugated to a topoisomerase I inhibitor payload (an exatecan derivative) via a tetrapeptide-based cleavable linker, can be an effective treatment for tamoxifen-resistant (TMR) ER+ BC. After first assessing HER3-DXd as a single agent, we sought to identify a synergistic partner to maximize HER3-DXd’s antitumor activity in HER3+/ER+ TMR BC. Methods: Whole-genome high-throughput siRNA screening was performed to identify targets whose inhibition enhances HER3-DXd’s antitumor efficacy. The antitumor effects of HER3-DXd plus the synergistic partners were assessed using a soft agar colony formation assay and a clonogenic assay in TMR HER3+/ER+ MCF7 and T47D BC cells. Treatment effects on cell cycle distribution, apoptosis, and expression of proteins that regulate cell cycle progression were assessed by flow cytometry, annexin V-PE and 7-AAD staining, and Western blotting, respectively. Results: HER3-DXd inhibited the anchorage-independent growth of HER3+/ER+ cells by &amp;gt;50% at 5 nM and their colony formation at 5-25 nM (P&amp;lt;0.05). To maximize HER3-DXd’s antitumor efficacy, we performed high-throughput siRNA screening and identified ATR, CD247, RAB7A, UPK3A, ROCK2, SLC29A1, and WNT7A as potential synergistic targets. Among these targets, inhibiting ATR with siRNA or BAY1895344 showed the most synergistic effect with HER3-DXd in 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. The combination of HER3-DXd plus BAY1895344 reprogrammed cell cycle progression from G2/M arrest to G1 arrest by inhibiting both ATR/Chk1/cyclin A2/CDK2 and ATR/Chk1/cyclin E/CDK2 signaling. The combination also reduced expression of H2AX, an ATR substrate that contributes to DNA repair, but increased that of γH2AX, indicating the induction of DNA damage. HER3-DXd and BAY1895344 synergistically inhibited growth of HER3+/ER+ BC cells by inducing apoptosis. Conclusion: The combination of HER3-DXd plus ATR inhibitors has therapeutic potential for overcoming tamoxifen resistance in HER3+/ER+ BC. We are currently validating the synergy in endocrine-resistant ER+ BC xenograft models. Citation Format: Xuemei Xie, Jangsoon Lee, Jon A. Fuson, Huey Liu, Young Jin Gi, Pang-Dian Fan, Kumiko Koyama, Debu Tripathy, Naoto T. Ueno. Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB088.

Abstract 2016: Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models

Abstract Introduction: Antibody-drug Conjugates (ADCs) targeting differentially expressed tumor cell-surface antigens show robust clinical activity in several solid tumor cancers, but none is yet available for osteosarcoma. Due to the high unmet medical need in this indication, there is an urgent requirement to identify respective osteosarcoma cell-surface antigens and evaluate the antitumor activity of an ADC that can deliver tailored cytotoxic payloads to osteosarcoma tumors expressing these targets. Methods: We used an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. uPARAP (MRC2) was found to be enriched in osteosarcoma at both protein and mRNA levels. The cell-surface expression of uPARAP (MRC2) was further validated by IHC and flow cytometry with osteosarcoma cell lines, PDX models, and patient tumor tissue microarray. As a proof of concept, three MRC2 targeted ADCs with different Microtubulin- and alkylating agent types of payloads were tested in 8 osteosarcoma PDX models. uPARAP (MRC2) ADC, control ADC, or vehicle control was administered IV to mice harboring flank tumors at a dose of 3mg/kg, weekly X 3 (ADCE-003), 2mg/kg on Day 1 (ADCE-010), and 10mg/kg weekly X 2 (ADCE-011). EFS for treatment (T) and control (C) groups, minimum relative tumor volume (minRTV), and objective response measures were analyzed. Results: Western blotting confirmed the expression of uPARAP (MRC2) in 8 osteosarcoma cell lines and 8 PDX models. Flow cytometry further validated cell-surface localization and expression levels of uPARAP (MRC2) in 7 osteosarcoma cell lines. We then performed immunohistochemistry (IHC) staining using an osteosarcoma tissue microarray from 37 patients and 19 PDX models. uPARAP (MRC2) was expressed in 97% of the patient samples and 95% of the PDXs. 51% of the patient samples and 39% of the PDXs had an overall H-score of 100 or higher. Mice tolerated ADCs well with minimal toxicity. All 3 ADCs significantly prolonged EFS in 6/8 osteosarcoma models. Complete response (CR) or maintained CR were observed in 2 models in ADCE-010 and ADCE-011 groups, respectively. Conclusions: uPARAP (MRC2) is highly expressed in most osteosarcoma samples, which makes it a viable target for respective antigen targeting ADC therapies. Three uPARAP (MRC2) targeting ADCs showed antitumor activities in osteosarcoma preclinical models which warrant further investigation of uPARAP targeting ADC therapies for osteosarcoma. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana Lazcano, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Niels Behrendt, Christoffer F. Nielsen, Lars H. Engelholm, Richard Gorlick. Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2016.

A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody–drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure–activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate—to our knowledge for the first time—the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.