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Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) and stem cell gene therapy require conditioning to ablate the recipient’s hematopoietic stem cells (HSC) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities, resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+tumor models. These data provide proof-of-concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSC, with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late-effects of these procedures which would greatly increase their applicability.

MYC-over expressing lymphomas display inherent resistance to standard chemoimmunotherapy regimens and are characterised by genomic instability and constitutive activation of the DNA damage response (DDR) pathway.

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19− cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ(DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situhybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situhybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

Jabeen: ADC Therapeutics: Current Employment. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Introduction.CD22 is a cell surface marker expressed by the vast majority of normal and neoplastic B-cells. ADCT-602 is an antibody drug conjugate (ADC) composed of Emab-C220, an engineered version of the anti-CD22 humanized IgG1 antibody epratuzumab, site-specifically conjugated to SG3249, which includes the DNA minor groove crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199 linked to the antibody via a protease-cleavable linker (Zammarchi et al, ASH 2016). ADCT-602 is currently being tested in a phase I/II clinical trial (NCT03698552) in recurrent or refractory B-cell acute lymphoblastic leukemia (B-ALL) patients. Here, we assessed its in vitroanti-lymphoma activity, also exploring for potential biomarkers and mechanisms of resistance.

Camidanlumab tesirine (ADCT-301) is an antibody-drug conjugate (ADC) comprised of HuMax®-TAC, a monoclonal antibody directed against human CD25, conjugated to the pyrrolobenzodiazepine dimer payload tesirine[1]. Currently, camidanlumab tesirine is being evaluated in a pivotal Phase 2 clinical trial in patients with relapsed or refractory Hodgkin lymphoma (HL) (NCT04052997) and in a Phase 1b clinical trial in patients with advanced solid tumors (NCT03621982). In pre-clinical studies, camidanlumab tesirine demonstrated strong and durable single agent activity in CD25-expressing lymphoma xenograft models[1] and in vitroit synergised with selected targeted agents[2]. Moreover, CD25-ADC, a mouse CD25 cross-reactive surrogate of camidanlumab tesirine, induced potent anti-tumor immunity against established syngeneic solid tumor models by depleting CD25-positive tumor-infiltrating T regulatory cells (Tregs) and it showed synergistic activity when combined with PD-1 blockade[3].

Overexpression of the MYC oncogene is a frequent feature of diffuse large B-cell lymphoma (DLBCL) being associated with poor prognosis following standard R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, Prednisone) chemoimmunotherapy. Since MYC expression is associated with overactivation of the DNA damage response (DDR), targeting DDR pathways with selective small molecule inhibitors could be a promising strategy to circumvent the inherent resistance to exogenous DNA damage proper of MYC-positive DLBCL. Loncastuximab tesirine-lpyl (here abbreviated as Lonca) is an antibody-drug conjugate (ADC) composed of a humanized anti-CD19 antibody conjugated to a potent DNA-crosslinking pyrrolobenzodiazepine dimer toxin.

Loncastuximab tesirine-lpyl (formerly ADCT-402) is an antibody-drug conjugate (ADC) comprising a humanised anti-CD19 monoclonal antibody conjugated to the pyrrolobenzodiazepine (PBD) dimer-based payload tesirine. Once bound to CD19 on the cell membrane, loncastuximab tesirine is rapidly internalised and the released PBD dimer warhead causes interstrand DNA crosslinks which ultimately trigger cell death. Pre-clinically, loncastuximab tesirine has shown potent and specific anti-tumor activity in lymphoma models both as single agent and in combination with other approved drugs, like venetoclax, idelalisib and bendamustine (Zammarchi, Corbett et al. 2018, Tarantelli, Spriano et al. 2019). Loncastuximab tesirine has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL) and it is currently being tested in multiple clinical trials, either as monotherapy or in combination with other anti-lymphoma drugs.

Sachini: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Jabeen: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. van Berkel: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Zammarchi: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Zammarchi: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. van Berkel: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Pileri: NANOSTRING: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD; CELGENE: Other: ADVISORY BOARD. Tarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD. Derenzini: TG-THERAPEUTICS: Research Funding; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; BEIGENE: Other: ADVISORY BOARD; TAKEDA: Research Funding; ADC-THERAPEUTICS: Research Funding.

Zammarchi: ADC Therapeutics: Employment. Van Berkel:ADC Therapeutics: Research Funding. Melnick:Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy. Luger:Celgene: Research Funding; Cyslacel: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Agios: Honoraria; Ariad: Research Funding; Biosight: Research Funding; Kura: Research Funding; Onconova: Research Funding; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria. Meffre:AbbVie: Consultancy, Other: Grant. Weinstock:Celgene: Research Funding.

Background:Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through b- and g-, but not a-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear.

Forman: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Weinstock:Genentech/Roche, Monsanto: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Travera: Equity Ownership; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties. Uzel:Novartis: Research Funding.

Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells.

Background and hypothesis:B cells critically depend on continuous survival and proliferation signals from a functional B cell receptor (BCR). In >50% of B cell malignancies, the tumor clone is driven by an oncogenic BCR-mimic. Oncogenic mimics of BCR-signaling include BCR-ABL1 (Ph+ALL) and other ABL1-fusion genes (Ph-like ALL), viral oncoproteins (e.g. EBV; KSHV), BRAF- (hairy cell leukemia) and NF-κB-pathway lesions (Hodgkin's lymphoma, primary mediastinal B cell lymphoma). B cell tumors driven by oncogenic BCR-signaling represent a highly heterogeneous group of various cellular origins, genetic lesions and divergent clinical outcomes. Despite this heterogeneity, our correlative analyses showed that these B cell malignancies are uniformly characterized by high expression levels of CD25.

Zammarchi: ADC Therapeutics: Employment. Corbett:ADC Therapeutics: Research Funding. Adams:Medimmune: Employment. Mellinas-Gomez:Medimmune: Employment. Tyrer:Medimmune: Employment. Dissanayake:ADC Therapeutics: Employment. Sims:ADC Therapeutics: Employment. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. Willimas:MedImmune: Employment. Howard:MedImmune/ ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hartley:Spirogen Ltd/ADC Therapeutics: Employment, Equity Ownership, Research Funding. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

We previously described the development of hLL2-PBD, an ADC composed of the humanized monoclonal antibody epratuzumab (hLL2) against human CD22, stochastically conjugated to SG3249, a cathepsin B-cleavable valine-alanine PBD payload. SG3249, also known as tesirine, is a potent and clinically validated PBD payload currently being employed in the clinic in three ADCs: the CD25-targeted ADCT-301, the CD19-targeted ADCT-402, both for the treatment of lymphoma and leukemia, and the DDL3-targeted rovalpituzumab tesirine for the treatment of small cell lung cancer. hLL2-PBD demonstrated potent and specific in vitroand in vivoactivity in the CD22-positive human Burkitt's lymphoma-derived cell lines Ramos, Daudi and WSU-DLCL2. Here, we describe the development and pre-clinical characterization of hLL2-cys-PBD, an ADC composed of an engineered version of epratuzumab (hLL2-cys), site-specifically conjugated to the PBD payload tesirine. The average drug to antibody ratio of hLL2-cys-PBD is 1.74.

Zammarchi: ADC Therapeutics: Employment. Williams:Spirogen/Medimmune: Employment. Adams:Spirogen/Medimmune: Employment, Equity Ownership. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. D'Hooge:Spirogen/Medimmune: Employment, Equity Ownership. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Human CD19 antigen is a 95 kilodalton type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (Wang, Wei, & Liu, 2012). The role of CD19, both in health and disease, is well studied, and the therapeutic efficacy and safety of CD19 modulation have been well defined over several decades (Scheuermann & Racila, 1995). In normal human tissue, expression of CD19 is limited to the various stages of B-cell development and differentiation (except plasma cells) and its expression is maintained on the majority of B-cell malignancies, including B-cell leukemia and non-Hodgkin lymphomas of B-cell origin. CD19 has rapid internalization kinetics and it is not shed into the circulation (Blanc et al., 2011; Gerber et al., 2009). All these features make CD19 an attractive target for the development of an ADC to treat B-cell malignancies.

ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitroand ex vivo,ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development.

Flynn: Medimmune: Employment. van Berkel:ADC Therapeutics Sarl: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics Sarl: Employment. Levy:Medimmune: Employment. Tiberghien:Medimmune: Employment, Patents & Royalties. Masterson:Medimmune: Employment. D'Hooge:Medimmune: Employment. Adams:Medimmune: Employment. Williams:Medimmune: Employment. Howard:ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties. Hartley:Medimmune: Employment, Equity Ownership, Patents & Royalties; ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties.

Interleukin-2 receptor-α (IL2R-α, CD25), one of a heterotrimer that makes up the IL2R, plays a key role in signal transduction pathways involved in the pathogenesis of autoimmunity and graft rejection (Burchill et al Immunol Lett 2007). In addition, the preponderance of CD25+ cells in hematological malignancies (Srivastava et al Leuk Lymphoma 1994) and the relationship between increased CD25 expression and poor prognosis (Yoshida et al PLoS One 2013) raise the possibility of using an anti-CD25 antibody to deliver a cytotoxin to these cells in patients. Clinical proof of concept for treatment of CD25-positive malignancies has previously been established using radio-immunoconjugates (Dancey et al Clin Cancer Res 2009) and immunotoxins (Kreitman et al J Clin Oncol 2000) utilising antibodies basiliximab and daclizumab.