Supplementary Data from Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody–Drug Conjugate (original) (raw)
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Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody-Drug Conjugate
Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patientderived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with proteaseactivated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.
In vivo effects of targeting CD79b with antibodies and antibody-drug conjugates
Molecular Cancer Therapeutics, 2009
Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at˜30 mg/kg (6,000 μg DM1/m 2 ) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of˜65% and˜94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclin-ical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases. [Mol
Biopharmaceutics & drug disposition, 2015
CD70 is a tumor necrosis factor (TNF)-like type II integral membrane protein that is transiently expressed on activated T- and B-lymphocytes. Aberrant expression of CD70 was identified in both solid tumors and haematologic malignancies. BMS-936561 (αCD70_MED-A) is an antibody-drug conjugate composed of a fully human anti-CD70 monoclonal antibody (αCD70) conjugated with a duocarmycin derivative, MED-A, through a maleimide-containing citrulline-valine dipeptide linker. MED-A is a carbamate prodrug that is activated by carboxylesterase to its active form, MED-B, to exert its DNA alkylation activity. In vitro serum stability studies suggested the efficiencies of hydrolyzing the carbamate-protecting group in αCD70_MED-A followed a rank order of mouse > rat > monkey > dog ~ human. Pharmacokinetics of αCD70_MED-A was evaluated in mice, monkeys, and dogs after single intravenous doses. In mice, αCD70_MED-A was cleared rapidly, with no detectable exposures after 15 min following dos...
Data from Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12
Purpose: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities.Experimental Design: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities.Results: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12β, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed simila...
Optimal design, anti-tumour efficacy and tolerability of anti-CXCR4 antibody drug conjugates
Scientific Reports, 2019
Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4 + cancer cells in solid tumours, but showed limited toxicity to normal CXCR4 + tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression. The discovery of CXCR4 as a co-receptor for T-tropic HIV-1 variants prompted a wealth of research into its biology and the development of CXCR4 small molecule inhibitors 1. Besides its function in HIV-1 infection, CXCR4 plays key roles during ontogenesis: chemotaxis of neural and vascular progenitors, migration of haematopoietic precursors from foetal liver to bone marrow and B-lymphocyte and myeloid cell development 2. As such, global knockouts of CXCR4 and its ligand CXCL12 are embryonic lethal 3-5. In adult tissues, CXCR4 is expressed in haematopoietic cells, adrenal gland, and kidney tubules 6-8 , whereas CXCL12 is a homeostatic chemokine, being expressed by mesenchymal stromal cells in many tissues 9. CXCL12/CXCR4 signalling has multiple functions in haematopoietic progenitor cells: maintenance of quiescence, retention in bone marrow and protection from oxidative stress 10-13. CXCR4 is also required for retention of granulocytic progenitors and neutrophils in the bone marrow 14. CXCR4 expression is often up-regulated in haematological malignancies 15 , and correlates with therapy resistance and poor prognosis in acute myelogenous leukaemia (AML) and non-Hodgkin lymphoma (NHL) 16-19. CXCR4 + haematological and solid tumour cells co-opt the role of CXCL12/CXCR4 in development and the "homing" of cancer cells to bone marrow is associated with therapy resistance and poor prognosis 20,21. Among
CXCL13 antibody for the treatment of autoimmune disorders
BMC Immunology, 2015
Background: Homeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity. Results: We developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)). Conclusions: We developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.
Development and Preclinical Characterization of a Humanized Antibody Targeting CXCL12
Clinical Cancer Research, 2013
The CXCL12/CXCR4 axis has been shown to play important roles in tumorigenesis and in a variety of other pathological conditions. Here we describe the development and characterization of a high-affinity humanized monoclonal antibody specific for CXCL12. The antibody had anti-tumor activity in multiple xenograft and orthotopic tumor models, as single agent or in combination with anti-VEGF antibodies. This antibody is suitable to test the hypothesis that targeting CXCL12 is a valid strategy to treat cancer and inflammatory diseases in humans.
mAbs, 2019
Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.