CD46 靶点调研报告

… identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted … its performance for immunoPET imaging in murine prostate cancer models. …

… of CD46-CAR T cells by suppressing the proliferation and liver metastasis in CD46 high-expressed CRC PDX mice. Collectively, our findings highlight that CD46-CAR T cell therapy …

CD46 is generally overexpressed in many human cancers, representing a prime target for CD46-binding adenoviruses (Ads). This could help to overcome low anti-tumoral activity by coxsackie-adenoviral receptor (CAR)-targeting cancer gene therapy viruses. However, because of scarce side-by-side information about CAR and CD46 expression levels in cancer cells, mixed observations of cancer therapeutic efficacy have been observed. This study evaluated Ad-mediated therapeutic efficacy using either CAR-targeting Ad5 or CD46-targeting Ad5/35 fiber chimera in bladder cancer cell lines. Compared with normal urothelia, bladder cancer tissue generally overexpressed both CAR and CD46. While CAR expression was not correlated with disease progression, CD46 expression was inversely correlated with tumor grade, stage, and risk grade. In bladder cancer cell lines, expression levels of CD46 and CAR were highly correlated with Ad5/35- and Ad5-mediated gene transduction and cytotoxicity, respectively. In a human EJ bladder cancer xenograft mouse model, with either overexpressed or suppressed CD46 expression levels, Ad5/35-tk followed by ganciclovir (GCV) treatment significantly affected tumor growth, whereas Ad5-tk/GCV had only minimal effects. Overall, our findings suggest that bladder cancer cells overexpress both CAR and CD46, and that adenoviral cancer gene therapy targeting CD46 represents a more suitable therapy option than a CAR-targeting therapy, especially in patients with low risk bladder cancers.

Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.

… CD46: structure, ligands, and functions in health and disease. Schematic of the key structural and functional features of CD46 including the CD46 … with perturbations in CD46 activity. …

… A potential toxicity concern is that an antibody-based therapy targeting CD46 may block complement inhibition and potentially induce atypical hemolytic uremic syndrome. However, our …

Background CD46 has been revealed to be a key factor in malignant transformation and cancer treatment. However, the clinical significance of CD46 in cervical cancer remains unclear, and this study aimed to evaluate its role in cervical cancer diagnosis and prognosis evaluation. Methods A total of 180 patients with an initial diagnosis of cervical cancer were enrolled at Taizhou Hospital of Zhejiang Province, China. The plasma levels of soluble CD46 (sCD46) and the expression of membrane-bound CD46 (mCD46) were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC), respectively. Results CD46 was found to be significantly upregulated in cervical cancer tissues vs. normal tissues, while no CD46 staining was detected in paired adjacent noncancerous tissues. CD46 staining was more pronounced in cancer cells than in stromal cells in situ (in tissues). Moreover, the plasma levels of sCD46 were able to some extent discriminate between cancer patients and healthy women (AUC=0.6847, 95% CI:0.6152–0.7541). Analysis of Kaplan–Meier survival curves revealed that patients with low CD46 expression had slightly longer overall survival (OS) than patients with high CD46 expression in the tumor microenvironment, but no significant difference. Univariate Cox regression analysis revealed that CD46 (P=0.034) is an independent risk factor for OS in cervical cancer patients. Conclusion The present study demonstrated that cervical cancer patients exhibit aberrant expression of CD46, which is closely associated with a poor prognosis, suggesting that CD46 plays a key role in promoting cervical carcinogenesis and that CD46 could serve as a promising potential target for precision therapy for cervical cancer.

CD46 is a complement inhibitor membrane cofactor which also acts as a receptor for various microbes, including species B adenoviruses (Ads). While most Ad gene therapy vectors are derived from species C and infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancer cells, resulting inefficient Ad-based therapeutics. Despite a limited knowledge on the expression status of many cancer cells, an increasing number of cancer gene therapy studies include fiber-modified Ad vectors redirected to the more ubiquitously expressed CD46. Since our finding from tumor microarray indicate that CD46 was overexpressed in cancers of the prostate and colon, fiber chimeric Ad5/35 vectors that have infection tropism for CD46 were employed to demonstrate its efficacy in colorectal cancers (CRC). CD46-overexpressed cells showed a significantly higher response to Ad5/35-GFP and to Ad5/35-tk/GCV. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and accordingly its cell killing. Injection of Ad5/35-tk/GCV caused much greater tumor-suppression in mice bearing CD46-overexpressed cancer xenograft compared to mock group. Analysis of CRC samples revealed that patients with positive CD46 expression had a higher survival rate (p=0.031), carried tumors that were well-differentiated, but less invasive and metastatic, and with a low T stage (all p<0.05). Taken together, our study demonstrated that species B-based adenoviral gene therapy is a suitable approach for generally CD46-overexpressed CRC but would require careful consideration preceding CD46 analysis and categorizing CRC patients.

… Given the lack of CAR expression on malignant gliomas and the need for alternative targets in gene therapy, we investigated the expression of CD46 on a variety of passaged and …

One of the most challenging aspects of cancer therapeutics is target selection. Recently, CD46 (membrane cofactor protein; MCP) has emerged as a key player in both malignant transformation as well as in cancer treatments. Normally a regulator of complement activation, CD46 is co-expressed as four predominant isoforms on almost all cell types. CD46 is highly overexpressed on a variety of human tumor cells. Clinical and experimental data support an association between increased CD46 expression and malignant transformation and metastasizing potential. Further, CD46 is a newly discovered driver of metabolic processes and plays a role in the intracellular complement system (complosome). CD46 is also known as a pathogen magnet due to its role as a receptor for numerous microbes, including several species of measles virus and adenoviruses. Strains of these two viruses have been exploited as vectors for the therapeutic development of oncolytic agents targeting CD46. In addition, monoclonal antibody-drug conjugates against CD46 also are being clinically evaluated. As a result, there are multiple early-phase clinical trials targeting CD46 to treat a variety of cancers. Here, we review CD46 relative to these oncologic connections.

… cancer patients in our cohort. To exploit the constitutive high levels of expression of CD46 on EOC cells, we evaluated the targeting … expressing CD46. We demonstrate a significant, …

Abstract Purpose: Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. Experimental Design: In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. Results: [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. Conclusions: Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Epidermal growth factor receptor (EGFR) is an effective target for those patients with metastatic colorectal cancers that retain the wild-type RAS gene. However, its efficacy in many cancers, including bladder cancer, is unclear. Here, we studied the in vitro effects of cetuximab monoclonal antibodies (mAbs) targeting EGFR on the bladder cancer cells and role of CD46. Cetuximab was found to inhibit the growth of both colon and bladder cancer cell lines. Furthermore, cetuximab treatment inhibited AKT and ERK phosphorylation in the bladder cancer cells and reduced the expression of CD46 membrane-bound proteins. Restoration of CD46 expression protected the bladder cancer cells from cetuximab-mediated inhibition of AKT and ERK phosphorylation. We hypothesized that CD46 provides protection to the bladder cancer cells against mAb therapies. Bladder cancer cells were also susceptible to cetuximab-mediated immunologic anti-tumor effects. Further, cetuximab enhanced the cell killing by activating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in bladder cancer cells. Restoration of CD46 expression protected the cells from both CDC and ADCC induced by cetuximab. Together, CD46 exhibited a cancer-protective effect against both direct (by involvement of PBMC or complement) and indirect cytotoxic activity by cetuximab in bladder cancer cells. Considering its clinical importance, CD46 could be an important link in the action mechanism of ADCC and CDC intercommunication and may be used for the development of novel therapeutic strategies.

CD46 is well known to be involved in diverse biological processes. Although several splice variants of CD46 have been identified, little is known about the contribution of alternative splicing to its tumorigenic functions. In this study, we found that exclusion of CD46 exon 13 is significantly increased in bladder cancer (BCa) samples. In BCa cell lines, enforced expression of CD46-CYT2 (exon 13-skipping isoform) promoted, and CD46-CYT1 (exon 13-containing isoform) attenuated, cell growth, migration, and tumorigenicity in a xenograft model. We also applied interaction proteomics to identify exhaustively the complexes containing the CYT1 or CYT2 domain in EJ-1 cells. 320 proteins were identified that interact with the CYT1 and/or CYT2 domain, and most of them are new interactors. Using an internal ribosome entry site (IRES)-dependent reporter system, we established that CD46 could regulate mRNA translation through an interaction with the translation machinery. We also identified heterogeneous nuclear ribonucleoprotein (hnRNP)A1 as a novel CYT2 binding partner, and this interaction facilitates the interaction of hnRNPA1 with IRES RNA to promote IRES-dependent translation of HIF1a and c-Myc. Strikingly, the splicing factor SRSF1 is highly correlated with CD46 exon 13 exclusion in clinical BCa samples. Taken together, our findings contribute to understanding the role of CD46 in BCa development.

Initially identified as a regulator of complement activation on host cells, the known roles of CD46 (membrane cofactor protein [MCP]) have expanded. We now know that this ancient molecule is expressed on almost all nucleated cells as a family of four predominant isoforms. It also is involved in human reproduction, modulation of T cell activation and immunoinflammatory effector functions, autophagy, and the newly identified intracellular complement system (complosome). CD46 is also known as a “pathogen” magnet, being a port of entry for at least seven bacteria and five viruses. Moreover, CD46 has recently emerged as a key player in cancer biology. Numerous studies provide evidence of the association among elevated CD46 expression, malignant transformation, and metastasizing potential. These features, along with its roles as pathogen receptor, have made CD46 a target for cancer therapeutics. Thus, modified viral vectors (such as strains of adenovirus and measles virus) targeting CD46 currently are being exploited against a wide range of cancers. Another oncologic treatment utilizes a CD46-targeting human mAb as an antibody-drug conjugate. Herein, we review CD46 and its “multiverse” of cancer interactions.

CD46 is a membrane-bound complement regulatory protein (mCRP) possessing a regulatory role with the complement system. CD46 protects the host cells from damage by complement. Expression of CD46 is also highly maintained in many cancers, including bladder cancers, and thus functions as a receptor for many cancer therapeutic viruses. In this study we report a unique role of CD46 as a progression factor of cancer cells in bladder cancers. Resulting data from a DNA microarray using CD46-altered HT1376 bladder cancers demonstrated a pool of target genes, including complement C3 α chain (C3α), matrix Gla protein (MGP), AFAP-AS1, follicular dendritic cell secreted protein (FDCSP), MAM domain containing 2 (MAMDC2), gamma-aminobutyric acid A receptor pi (GABRP), transforming growth factor, beta-induced (TGFBI), a family of cytochrome P450 (CYP24A1), sialic acid binding Ig-like lectin 6 (SIGLEC6), metallothionein 1E (MT1E), and several members of cytokeratins. Subsequent studies using quantitative RT-PCR and Western blot analyses confirmed CD46-mediated regulation of C3α, MGP, and keratin 13 (KRT13). MGP and KRT13 are known to be involved in cell migration and cancer cell metastasis. A cell migration assay demonstrated that CD46 enhanced migratory potential of bladder cancer cells. Taken all together, this report demonstrated that CD46 is generally overexpressed in bladder cancers and plays a unique role in the promotion of cancer cell migration. Further detailed studies are needed to be performed to clarify the action mechanism of CD46 and its application to cancer therapeutics.

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated ^212Pb, an in vivo generator of alpha-emitting ^212Bi and ^212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, ^212Pb-TCMC-YS5. We characterized ^212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of ^212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 0.74 MBq (20 µCi) ^212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (0.37 MBq or 10 µCi ^212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that ^212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment.

PURPOSE FOR46, a fully human antibody conjugated to monomethyl auristatin E, targets a tumor-selective epitope of CD46, which is overexpressed in metastatic castration-resistant prostate cancer (mCRPC). FOR46 demonstrates potent nonclinical activity in enzalutamide-resistant CRPC models. PATIENTS AND METHODS This was a phase I, first-in-human, dose escalation/expansion study in patients with progressive mCRPC after treatment with ≥one androgen signaling inhibitors (ClinicalTrials.gov identifier: NCT03575819). The starting dose of FOR46 was 0.1 mg/kg given intravenously every 3 weeks. The primary objective was to determine the maximally tolerated dose (MTD). Whole-blood mass cytometry (cytometry by time of flight) was used to characterize peripheral immune response and CD46 expression in CRPC tissue that underwent central pathology review. RESULTS Fifty-six patients were enrolled. Dose-limiting toxicities included neutropenia (n = 4), febrile neutropenia (n = 1), and fatigue (n = 1). The MTD was 2.7 mg/kg using adjusted body weight. The most common grade ≥3 adverse events across all dose levels were neutropenia (59%), leukopenia (27%), lymphopenia (7%), anemia (7%), and fatigue (5%). One grade 3 febrile neutropenia event was observed. There were no treatment-related deaths. In the efficacy evaluable subset (patients with adenocarcinoma treated with a starting dose ≥1.2 mg/kg, n = 40), the median radiographic progression-free survival was 8.7 months (range, 0.1-33.9). Fourteen of 39 evaluable patients (36%) achieved a PSA50 response. The confirmed objective response rate was 20% (5 of 25 RECIST-evaluable patients). The median duration of response was 7.5 months. Responders had a significantly higher on-treatment frequency of circulating effector CD8+ T cells. CONCLUSION FOR46 demonstrated encouraging preliminary clinical activity with a manageable safety profile. Targeting CD46 elicited an immune priming effect that was associated with clinical outcomes.

OBJECTIVE Multiple myeloma (MM) is an incurable B cell malignancy and novel therapeutics are urgently needed. Live attenuated measles virus (MV) has potent oncolytic activity against MM tumor xenografts. The virus is tumor selective and preferentially targets cells that express high levels of CD46 receptors. However, CD46 levels on MM have not previously been evaluated. In this study, we investigated the potential of CD46 as a target for MM therapy and correlated surface levels of CD46 on MM cells with their susceptibility to MV-induced cytopathic effects. MATERIALS AND METHODS CD46 expression on neoplastic plasma cells (PCs) and nonplasma cells (NPCs) from 38 MM patients was analyzed by flow cytometry and receptor numbers were quantitated using BD QuantiBRITE PE beads. RESULTS Results showed that malignant PCs expressed significantly higher levels of CD46 receptors compared to NPCs (p < 0.0001). The mean CD46 receptor numbers on PCs and NPCs were 49,130/cell and 7,340/cell, respectively. Potent cytopathic effects of extensive intercellular fusion were observed in measles-infected PCs but not in NPCs. The extent of MV-induced cytopathic effects of cell fusion correlated with CD46 expression levels on the MM cells. Normal plasma cells do not overexpress CD46 and colony-forming assays demonstrated that MV was not cytotoxic to normal bone marrow progenitor cells. CONCLUSION The present study establishes CD46 as a surface antigen that is expressed more abundantly on primary MM cells compared to normal hematopoietic cells of various lineages in the bone marrow, making CD46 a promising surface marker for targeted cytoreductive therapy of MM.

3001 Background: FOR46, a fully human antibody (ab) conjugated to monomethyl auristatin E (MMAE), targets a tumor selective epitope of CD46, which is highly expressed in mCRPC and treatment-emergent small cell neuroendocrine cancer (t-SCNC). CD46 is enriched in tumor cells upon treatment with androgen signaling inhibitors (ASI). Following dose escalation (Phase 1a), dose expansion was undertaken in 2 cohorts (Phase 1b): 1) Pts with de novo or t-SCNC and 2) pts with mCRPC without a t-SCNC component. Pts with adenocarcinoma enrolled in dose escalation and expansion are included in this analysis. Methods: Eligible pts had mCRPC, with progression on at least 1 ASI, with no prior chemotherapy for CRPC. Phase 1a pts received FOR46 0.1-3.0 mg/kg IV Q3 weeks (wks). The primary objectives in phase 1a were to assess adverse effects (AEs) and select the phase 1b dose; and in phase 1b to assess efficacy. For phase 1b, tumor biopsy in the CRPC setting for assignment to the 2 cohorts was required. CD46 expression was not required for inclusion in the expansion cohort, but was evaluated using a non-epitope specific CD46 polyclonal ab. Histology and CD46 expression were centrally reviewed. Results: Thirty-three pts were enrolled in phase 1a and 10 in phase 1b (including 6 treated in ph1a at the expansion dose or higher). Overall, 36 pts were treated at doses > 1.2 mg/kg. Following excess toxicity in pts with body mass indices > 30 (3 of 3 with Gr 4 neutropenia and 1 of 3 with Gr 3 fatigue at 2.4 mg/kg), further dosing was calculated using adjusted body weight (AJBW) rather than actual weight, allowing escalation to 3.0 mg/kg. The 2.7 mg/kg dose by AJBW was determined to be the MTD and phase 1b dose. The most common AEs at the 2.7 mg/kg dose were neutropenia (77% Gr 3 or 4), infusion reactions (37%, all < Gr 2), fatigue (31%, all < Gr 2) and peripheral neuropathy (24%, all < Gr 2)). Fourteen of 31 evaluable pts (45.2%) at > 1.2 mg/kg achieved a PSA50 response with 10 (32.3%) confirmed. Five pts were not evaluable for PSA response; 3 had no post-baseline PSA and 2 had baseline PSA < 1 ng/mL. The median duration of confirmed PSA50 response is >16 wks (range 6-48+ wks, with 4 ongoing at 12, 24, 25 and 48 wks). 18 pts had measurable lesions; 8 of 18 (44.4%) had tumor regression, with 4 (22.2%) confirmed partial responses (PR). The median duration of response is > 14 wks (range 9 -31+ weeks with 2 ongoing at 13 and 31 wks). Eight pts were evaluable for CD46 expression with a median H-score of 245 (range 0-300). Two pts with PRs had H-scores of 15 and 300; 4 with confirmed PSA50 had H-scores of 10, 15, 40 and 300. Conclusions: FOR46, a novel ADC targeting CD46, demonstrates clinical activity in mCRPC pts, with an acceptable safety profile, similar to other MMAE-containing ADCs. FOR46 merits further investigation in pts with mCRPC, alone and in combination with agents that enhance CD46 expression. Clinical trial information: NCT03575819.

Simple Summary Multiple myeloma (MM) is incurable, implying that the disease cells are inherently resistant to current agents and inevitably lead to relapse. One strategy to improve the treatment paradigm is to pursue novel drug targets that are associated with relapsed disease. Towards this end, we developed a novel antibody–drug conjugate (ADC) that targets the cell surface complement inhibitor CD46. Here, we study the potential of this new agent to affect disease initiation. The gene encoding for CD46 is amplified on chromosome 1q in the majority of relapsed myeloma patients, as well as cells containing stem-like aldehyde dehydrogenase activity. We demonstrate the curative potential of CD46–ADC via its ability to abrogate disease engraftment of primary MM cell xenografts. In combination with a recent clinical trial, this study supports the continued study of CD46 as a therapeutic target in MM. Abstract 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.

… CD46-targeting antibody drug conjugate and 225 Ac-based alpha particle therapy agents … a treatment strategy targeting CD46 using simultaneous antibody-drug conjugate (ADC) and …

… antibody-drug conjugate targeting CD46 conjugated to monomethyl auristatin F (MMAF) anti-tubulin payload (CD46-… CD46 and primary cells with 1q+ are more sensitive to CD46-ADC. …

5066 Background: FOR46 is an MMAE-containing antibody-drug conjugate (ADC) targeting a tumor-specific conformational epitope on the extracellular domain of CD46 on prostate cancer and other cancer tissues in a lineage independent fashion. A prior phase 1, first-in-human trial of FOR46 demonstrated encouraging preliminary activity in mCRPC. In pre-clinical models, androgen receptor blockade with enzalutamide enhances CD46 epitope expression and achieves additive activity in combination with the CD46 ADC. We sought to evaluate the combination of FOR46 plus enza in mCRPC patients (pts). Methods: Pts with mCRPC with progression on ≥ 1 androgen receptor pathway inhibitor (ARPI) were enrolled. No prior chemotherapy for mCRPC was allowed. A 3+3 dose escalation design was utilized with a starting dose of FOR46 of 1.8 mg/kg adjusted body weight (ABW) in combination with enza 160 mg/day. Dose escalation was explored with and without prophylactic granulocyte colony-stimulating factor (G-CSF) support. The primary endpoint was determination of the maximally tolerated dose (MTD) of FOR46 in combination with enza. A baseline CD46-directed PET imaging probe utilizing the same antibody backbone as FOR46 (89Zr-DFO-YS5) was obtained in a subset of pts. Results: Seventeen pts were enrolled. Median age was 71 years (range 58 – 91) and median PSA was 43.8 ng/mL (range 2.1 – 379.6) at study entry. Twelve pts (70.6%) had ≥ 2 lines of prior ARPI. The median duration of treatment was 5.0 months (range 1– 18). Dose-limiting toxicities (DLTs) included grade 4 neutropenia at a dose of 2.1 mg/kg without G-CSF (n = 2), grade 4 hyponatremia at 2.4 mg/kg (n = 1), and grade 3 elevated transaminases at 2.4 mg/kg (n = 1). The MTD of FOR46 was established at 2.1 mg/kg ABW, with primary G-CSF prophylaxis, in combination with enza 160 mg/day. Grade ≥ 3 treatment-related adverse events (trAEs) were observed in 29.4% of pts. The most common trAEs of any grade were fatigue (n = 11, 64.7%), peripheral sensory neuropathy (n = 8, 47.1%), elevated transaminases (n = 3, 17.6%), alopecia (n = 4, 23.5%), decreased appetite (n = 7, 41.2%), neutropenia (n = 3, 17.6%), and weight loss (n = 3, 17.6%). Grade 2 neuropathy was observed in 2 patients (11.8%). Preliminary anti-tumor activity was observed with PSA declines in 13/16 (81.3%) of evaluable pts, and a disease control rate (stable disease ≥ 6 months) of 41.2%. 89Zr-DFO-YS5 demonstrated tumor uptake on whole body PET with pharmacokinetics typical for an IgG radiopharmaceutical. Conclusions: In combination with enza, the established MTD of FOR46 with primary G-CSF prophylaxis was safe and demonstrated preliminary evidence of efficacy. 89Zr-DFO-YS5 PET demonstrates tumor-specific uptake and will be employed in the ongoing Phase 2 portion of the study as a potential predictive biomarker of response. Clinical trial information: NCT05011188 .

Background: Multiple myeloma (MM) is incurable by standard approaches, with relapse and development of treatment resistance inevitable in all patients. We previously identified a panel of novel macropinocytosing human monoclonal antibodies against CD46 by phage antibody library display and optimized a lead antibody for targeted drug delivery. Antibody-drug conjugates (ADCs) have recently seen proof-of-concept clinical success in Hodgkin lymphoma and breast cancer, but none is yet FDA-approved for MM. The CD46 gene is located on the long arm of chromosome 1 (1q32.2), 50Mbp from a FISH probe clinically used to identify high-risk MM and which may provide a surrogate biomarker for CD46 as a therapeutic target. Methods:We covalently conjugated the monomethyl auristatin F (MMAF) toxin to our anti-CD46 antibody via a lysosomal protease sensitive valine-citrulline linker (hereafter referred to as CD46-ADC). High Performance Liquid Chromatography analysis with hydrophobic interaction chromatography of the final conjugate showed an average drug per antibody of 3.3. CD46-ADC was evaluated for cytotoxicity in vitro in MM cell lines, in vivo with cell line xenografts in NSG mice, and ex vivo in MM patient bone marrow (BM)aspirate samples. To assess in vivo toxicity, CD46-ADC treatment was administered to transgenic mice that express the human CD46 gene under its native promoter. Results: CD46 was highly expressed on the cell surface of all 18 MM cell lines tested, and was upregulated on MM1.S cells co-cultured with the BM stromal cell line HS5. In BM aspirate samples, CD46 was highly expressed on MM cells in 100% (n=25) patients evaluated. By quantitative flow cytometry in 10 patients, the CD46 cell surface antigen density was significantly higher in patient MM cells with 1q21 gain (1q+) than those with normal 1q21 copy number (p=0.032) (Fig 1A). In patients with amp1q21 the mean CD46 antigen density on MM cells was 313,190 (SEM 68,849), compared to patients with normal 1q21 where it was 121,316 (SEM 28,352) (Fig 1A). In contrast, CD46 antigen density on normal donor (n=3) BM hematopoietic cell populations was low (antigen density range 8,443 - 23,772). Of note, higher CD46 antigen density was present on monocytes (mean 58,320, SEM 6,874) and granulocytes (mean 54,439, SEM 10,688) relative to the other populations (Fig 1B). CD46-ADC potently inhibited proliferation in all 14 MM cell lines tested (EC50 range of 150 pM - 5 nM) (Fig 1C). On BM stromal cells, CD46-ADC had EC50 &gt;100 nM for patient-derived BM61 (generated via culture of CD138-negative BM) cells and no effect on HS5 cells in concentrations tested up to 150 nM. CD46-ADC eliminated MM growth in two orthometastatic xenograft models. In one model, MM1.S cell line xenografts expressing firefly luciferase grown in NSG mice were treated once every 3-4 days at either 4 mg/kg or 0.8 mg/kg for 4 injections, or with a single dose of 4 mg/kg (Fig 2A). Control groups were treated with vehicle, nonbinding ADC or naked antibody (CD46-mAb). CD46-ADC 4 mg/kg (4 dose) eliminated bioluminescent activity throughout the duration of the study (Fig 2B), and all mice survived to study discontinuation (Fig 2C). The single dose and low dose groups showed elimination of bioluminescence, but all mice relapsed (Fig 2B-C). In patient BM aspirate samples, CD46-ADC induces apoptosis and cell death in primary MM cells ex vivo (EC50 &lt;10 nM), but did not affect the viability of non-tumor mononuclear cells (MNCs). For in vivo toxicity study, human CD46 transgenic mice were treated with a single IV bolus injection of 6 mg/kg CD46-ADC and showed no body weight loss or overt side effects for 14 days. At study discontinuation (day 14), histologic analysis of major organs showed no notable tissue damage. Conclusion: We have identified a novel functional antigen, CD46, for ADC targeting of MM, with unique potential for high-risk and relapsed/refractory disease that has genomic amplification at the CD46 gene locus and are in dire need of therapy. The novel CD46-ADC is highly potent and selective in eliminating MM cells (cell lines and primary tumor cells) in preclinical models. CD46 genomic gain on chromosome 1q correlates with antigen amplification, andindentifies a potential biomarker based on a clinical FISH test that can be used for patient stratification. Thus, our study could lead directly to the application of a novel ADC therapeutic for treating MM. Disclosures Aftab: Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding; CytomX: Research Funding; Cleave Biosciences, Inc.: Research Funding. Wiita:Onyx Pharmaceuticals: Research Funding; Omniox, LLC: Research Funding; Cleave Biosciences: Research Funding; Quadriga Biosciences: Research Funding. Wolf:Celgene: Honoraria; Telomere Diagnostics: Consultancy; Takeda: Honoraria; Amgen: Honoraria; Pharmacyclics: Honoraria. Martin:Sanofi: Research Funding; Amgen: Research Funding.

ABSTRACT Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

The therapeutic potential of anticancer antibodies is limited by the resistance of tumor cells to complement‐mediated attack, primarily through the over‐expression of membrane complement regulatory proteins (mCRPs: CD46, CD55 and CD59). Trastuzumab, an anti‐ HER2 monoclonal antibody, approved for the treatment of HER2‐positive breast and gastric cancers, exerts only minor complement‐mediated cytotoxicity (CDC). Pertuzumab is a novel anti‐HER2 monoclonal antibody, which blocks HER2 dimerization with other ligand‐activated HER family members. Here, we explored the complement‐mediated anti‐tumor effects of trastuzumab and pertuzumab on HER2‐positive tumor cells of various histological origins.Delivery of chemically stabilized anti‐mCRP siRNAs using cationic lipoplexes, AtuPLEXes, to HER2‐over‐expressing BT474, SK‐BR‐3 (breast), SKOV3 (ovarian) and Calu‐3 (lung) cancer cells reduced mCRPs expression by 85–95%. Knockdown of individual complement regulators variably led to increased CDC only upon combined treatment with trastuzumab and pertuzumab. The combined down‐regulation of all the three regulators augmented CDC by 48% in BT474, 46% in SK‐BR‐3 cells, 78% in SKOV3 cells and by 30% in Calu‐3 cells and also increased complement‐induced apoptosis and caspase activity on mCRP neutralized tumor cells. In addition, antibody‐induced C3 opsonization of tumor cells was significantly enhanced after mCRP silencing and further augmented tumor cell killing by macrophages.Our findings suggest that siRNA‐induced inhibition of complement regulator expression clearly enhances complement‐ and macrophage‐mediated anti‐tumor activity of trastuzumab and pertuzumab on HER2‐positive tumor cells. Thus – if selectively targeted to the tumor – siRNA‐induced inhibition of complement regulation may serve as an innovative strategy to potentiate the efficacy of antibody‐based immunotherapy.

BACKGROUND: One reason for the failure of monoclonal antibody (mab) trials in most cancer patients might be the presence of complement resistance factors that inhibit complement dependent cytotoxicity (CDC) and the release of inflammatory mediators (e.g., anaphylatoxins). METHOD: We have determined the expression of CD46, CD55, and CD59 in five gastric, three colon, and seven pancreatic human cancer cell lines by immunostaining. The complement activating properties of mabs and conjugates with cobra venom factor (CVF) were studied in a 51Cr-release toxicity assay and in an ELISA to determine the release of C3a. RESULT: Virtually all tumor cell lines strongly expressed CD46, CD55, and CD59, except KATOIII gastric cancer cells (CD55 and CD59 negative). In accordance with other studies we could confirm that expression of CD55 and CD59 inhibits a complement activation by mabs. Whereas 17-1A was able to induce a cytotoxic complement activation on KATOIII cells, neither a CDC nor an anaphylatoxin release (C3a) was observed on MKN28 cells (strong expression of CD55 and CD59). Conjugation with CVF, a strong activator of the alternative pathway of complement, could partially restore the complement activation by mabs. A 17-1A-CVF conjugate, although still nontoxic, induced the release of the anaphylatoxin C3a on both cell lines. The same observations were made in PancTuI pancreatic cancer cells treated with a conjugate of the mab CA19-9 and CVF. CONCLUSIONS: Our study shows that complement resistance is a frequent event in gastrointestinal cancer, limiting the potential of monoclonal antibodies. Mabs, when conjugated with CVF, partially retain complement activating properties by releasing C3a, which in vivo will support a cellular immune response.

… CD46 becomes proteolytically modified on cell membranes. We observed that tumor cells liberated intact 60–65 kDa forms of CD46 … Furthermore, soluble CD46 (55–60 kDa) containing …

… CD46 on breast tumours using a rabbit polyclonal anti-CD46 … with primary operable breast cancer diagnosed between 1987 … 507/510 (99.4%) of breast tumours expressed CD46. Strong …

The therapeutic potential of membrane complement regulatory protein (mCRP)-neutralizing antibodies is unsatisfactory, which perhaps lies in the complex role of mCRPs in tumor occurrence and development. As a member of the mCRPs, CD46 is a transmembrane protein with a cytoplasmic domain and is implicated more in the control of the alternative complement pathway than of the classical complement pathway. Growing evidence has revealed that both the CD46 signaling pathway and microRNAs (miRNAs) play an important role in the development and progression of hepatocellular carcinoma (HCC). In the present study, we analyzed mCRP expression in different tumor tissues by employing western blotting and qPCR. To address the potential role of miRNAs in CD46 signaling, we set out to profile miRNA expression in CD46-overexpressed and -silenced HepG2 cell lines. Furthermore, bioinformatic analysis was performed to identify downstream targets of CD46 signaling. We found that the levels of CD46 expression in HCC tissues were significantly higher compared to that in the adjacent normal tissues. After complement-related gene expression profiling and unsupervised hierarchical clustering analysis of 10 HCC tissues, a total of 37 miRNAs showed significantly different expression levels before and after CD46 expression change. By bioinformatic analysis, we identified let-7b and miR-17 as downstream targets of CD46 signaling, and that the expression levels of let-7b and miR-17 were negatively correlated with that of CD46 in HepG2 cells. The present study suggests that CD46 plays an important role in HCC carcinogenesis by regulating let-7b and miR-17.

… /ml of soluble membrane cofactor protein (MCP, CD46) whereas sera of >50% of the cancer … MCP purified by Immunoaffinlty chromatography from both normal and cancer patients' sera …

We have developed polyclonal and monoclonal antibodies against human membrane cofactor protein (MCP) to use as tools to investigate the functions of MCP on intact nucleated cells. Two human T cell lines, CEM and TALL, are CR1- and DAF-. Pretreatment of these cell lines with M177 and polyclonal anti-MCP, which inhibit cofactor activity almost completely, resulted in effective C3 deposition immediately following addition of these cells to Mg2+/EGTA/human sera. The deposited C3 remained expressed partly on the cell surface and most of them were gradually converted to C3bi. Some of the deposited C3 were complexed with membrane proteins, since 140- and 250-kD bands became significantly accumulated on SDS-PAGE by treatment with the antibodies. We next tested whether these C3-coated cells were damaged by complement- mediated cytolysis. p18, an inhibitor of membrane attack complex (MAC) formation, was negative in TALL but positive in CEM. TALL was lysed efficiently only by treatment with the polyclonal anti-MCP, while CEM showed only slight lysis with the same treatment. Monoclonal antibodies to MCP, including M177, caused only minimal cell destruction. Based on these results, together with the fact that decay-accelerating factor (DAF) serves as a factor for preventing C3 attack on human cells, we conclude that MCP and DAF cooperatively protect host cells from C3 targeting and, in these T cell lines, MCP is sufficient for preventing C3 deposition even without DAF. After all, human cells undergo almost no autologous complement-mediated cytolysis if they express at least one of the functionally active inhibitors, MCP, DAF, or p18.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer (PCa) and other malignancies. We previously reported high CD46 expression in PCa and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. Our radiolabeled antibody retains binding efficacy and shows a high tumor to background ratio in PCa xenografts. Furthermore, we show that radiolabeled antibody was able to suppress the growth of cell-derived and patient-derived xenografts, including PSMA-positive and deficient models. Nephrotoxicity, not seen at low radioactive doses, is evident at higher radioactivity dose levels, likely due to redistribution of daughter isotope 213Bi. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46 targeted radioligand therapy in PCa.

We have developed a technology that depletes the complement regulatory protein (CRP) CD46 from the cell surface, and thereby sensitizes tumor cells to complement-dependent cytotoxicity triggered by therapeutic monoclonal antibodies (mAbs). This technology is based on a small recombinant protein, Ad35K++, which induces the internalization and subsequent degradation of CD46. In preliminary studies, we had demonstrated the utility of the combination of Ad35K++ and several commercially available mAbs such as rituximab, alemtuzumab, and trastuzumab in enhancing cell killing in vitro as well as in vivo in murine xenograft and syngeneic tumor models. We have completed scaled manufacturing of Ad35K++ protein in Escherichia coli for studies in nonhuman primates (NHPs). In macaques, we first defined a dose of the CD20-targeting mAb rituximab that did not deplete CD20-positive peripheral blood cells. Using this dose of rituximab, we then demonstrated that pretreatment with Ad35K++ reconstituted near complete elimination of B cells. Further studies demonstrated that the treatment was well tolerated and safe. These findings in a relevant large animal model provide the rationale for moving this therapy forward into clinical trials in patients with CD20-positive B-cell malignancies.

… and efficient target-mediated internalization to facilitate the … In this study, median OS for EV and pembrolizumab was 31.5 … CD46 is a type I membrane protein that binds to complement …

The lack of success in prostate cancer from immune checkpoint inhibitors, which is likely multifactorial, has led to the development and investigation of a number of other novel immunotherapeutic techniques, including antibody–drug conjugates, T-cell redirected bispecific therapies, cancer vaccines and chimeric antigen receptor T-cell therapies. Prostate-specific membrane antigen (PSMA) is a tumour-associated antigen (TAA) that is highly expressed in metastatic prostate cancer and has been validated as an effective target for radionuclide treatment. But while PSMA has thus far been the ‘front runner’ target for these novel immunotherapeutic techniques, it may not be the ideal target for immunotherapy and there are other potential targetable TAAs that will require further exploration. This review will focus on these various PSMA-directed therapies, as well as other potential targets for immunotherapy beyond PSMA.

… potential for gene therapy and immunotherapy approaches (12)… target cell (CHO-SLAM or 293T cells for SLAM- or CD46-… H with CD46-expressing cells induces internalization of this …

… successful antibody-based cancer immunotherapy. To avoid a potential … -lipoplexes leads to internalization of siRNA-lipoplexes via … Targeted knockdown of individual CD46 and CD55 …