Antibody-based Therapies Research Articles

Conjugation of glucosylated polymer chains to checkpoint blockade antibodies augments their efficacy and specificity for glioblastoma.

Because of the blood-tumour barrier and cross-reactivity with healthy tissues, immune checkpoint blockade therapy against glioblastoma has inadequate efficacy and is associated with a high risk of immune-related adverse events. Here we show that anti-programmed death-ligand 1 antibodies conjugated with multiple poly(ethylene glycol) (PEG) chains functionalized to target glucose transporter 1 (which is overexpressed in brain capillaries) and detaching in the reductive tumour microenvironment augment the potency and safety of checkpoint blockade therapy against glioblastoma. In mice bearing orthotopic glioblastoma tumours, a single dose of glucosylated and multi-PEGylated antibodies reinvigorated antitumour immune responses, induced immunological memory that protected the animals against rechallenge with tumour cells, and suppressed autoimmune responses in the animals' healthy tissues. Drug-delivery formulations leveraging multivalent ligand interactions and the properties of the tumour microenvironment to facilitate the crossing of blood-tumour barriers and increase drug specificity may enhance the efficacy and safety of other antibody-based therapies.

Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

Polyclonal alpaca antibodies protect against hantavirus pulmonary syndrome in a lethal Syrian hamster model

The use of antibody-based therapies for the treatment of high consequence viral pathogens has gained interest over the last fifteen years. Here, we sought to evaluate the use of unique camelid-based IgG antibodies to prevent lethal hantavirus pulmonary syndrome (HPS) in Syrian hamsters. Using purified, polyclonal IgG antibodies generated in DNA-immunized alpacas, we demonstrate that post-exposure treatments reduced viral burdens and organ-specific pathology associated with lethal HPS. Antibody treated animals did not exhibit signs of disease and were completely protected. The unique structures and properties, particularly the reduced size, distinct paratope formation and increased solubility of camelid antibodies, in combination with this study support further pre-clinical evaluation of heavy-chain only antibodies for treatment of severe respiratory diseases, including HPS.

Antibody-Based Therapy in AML: Antibody–Drug Conjugates and Bispecific Agents

<h2>Introduction</h2> Remissions following therapy for acute myeloid leukemia (AML) are often not durable, and conventional treatments can have substantial toxicity. The last few years have witnessed an emergence of promising therapies for patients with AML. Antibody-based therapies can target leukemic cells via a variety of mechanisms. Conjugation of antibodies with cytotoxic agents or alteration of antibody segments to allow concurrent activation of T cells have transformed and expanded the therapeutic potential of antibody-based treatment. Identification and characterization of antigens specific to AML cells have helped to identify targets for antibody-drug conjugate (ADC) therapy. In addition, bispecific T cell engagers (BiTE) and dual-affinity retargeting (DART) therapies are emerging as therapeutically effective targeted immune-based treatment for patients with AML. Incorporating the target antigen-binding domain of an antibody, these molecules then engage and active the human immune system against leukemic cells expressing that target.

Antibody Conjugates for Sarcoma Therapy: How Far along Are We?

Sarcomas are one of the most difficult type of cancer to manage and treat because of their extremely heterogeneous molecular and morphological features. Despite the progress made over the years in the establishment of standard protocols for high and low grading/staging sarcoma patients, mostly with chemotherapy and/or radiotherapy, 50% of treated patients experience relapse episodes. Because of this, in the last 20 years, new therapeutic approaches for sarcoma treatment have been evaluated in preclinical and clinical studies. Among them, antibody-based therapies have been the most studied. Immunoconjugates consist of a carrier portion, frequently represented by an antibody, linked to a toxic moiety, i.e., a drug, toxin, or radionuclide. While the efficacy of immunoconjugates is well demonstrated in the therapy of hematological tumors and more recently also of epithelial ones, their potential as therapeutic agents against sarcomas is still not completely explored. In this paper, we summarize the results obtained with immunoconjugates targeting sarcoma surface antigens, considering both preclinical and clinical studies. To date, the encouraging results obtained in preclinical studies allowed nine immunoconjugates to enter clinical trials, demonstrating the validity of immunotherapy as a promising pharmacological tool also for sarcoma therapy.

On the road to eliminating long-lived plasma cells-"are we there yet?"

Central to protective humoral immunity is the activation of B cells and their terminal differentiation into antibody-secreting plasma cells. Long-lived plasma cells (LLPC) may survive for years to decades. Such long-lived plasma cells are also responsible for producing pathogenic antibodies that cause a variety of challenges such as autoimmunity, allograft rejection, and drug neutralization. Up to now, various therapeutic strategies aimed at durably eliminating pathogenic antibodies have failed, in large part due to their inability to efficiently target LLPCs. Several antibody-based therapies have recently gained regulatory approval or are in clinical phases of development for the treatment of multiple myeloma, a malignancy of plasma cells. We discuss the exciting potential of using these emerging cancer immunotherapies to solve the antibody problem.

Improving Tumor Penetration of Antibodies and Antibody-Drug Conjugates: Taking Away the Barriers for Trojan Horses.

The high affinity of an antibody can result in restricted tumor penetration and heterogenous tumor distribution, with preferential binding of the antibody to tumor cells localized around tumor vasculature. This so-called "binding site barrier" effect limits the efficacy of antibody-based therapies like antibody-drug conjugates (ADC). In this issue, Bordeau and colleagues introduce an original approach to overcome this barrier through transient competitive inhibition of antibody-antigen binding. By coadministration of an anti-idiotypic anti-trastuzumab domain antibody as a competitive inhibitor, increased tumor penetration of trastuzumab as well as enhanced efficacy of the ADC ado-trastuzumab emtansine were observed in tumor-bearing miceSee related article by Bordeau et al., p. 4145.

Protecting the vulnerable: SARS-CoV-2 vaccination in immunosuppressed patients with interstitial lung disease

Protecting the vulnerable: SARS-CoV-2 vaccination in immunosuppressed patients with interstitial lung disease

Emergence of the E484K mutation in SARS-COV-2-infected immunocompromised patients treated with bamlanivimab in Germany.

BackgroundMonoclonal antibodies (mAb) have been introduced as a promising new therapeutic approach against SARS-CoV-2. At present, there is little experience regarding their clinical effects in patient populations underrepresented in clinical trials, e.g. immunocompromised patients. Additionally, it is not well known to what extent SARS-CoV-2 treatment with monoclonal antibodies could trigger the selection of immune escape viral variants.MethodsAfter identifying immunocompromised patients with viral rebound under treatment with bamlanivimab, we characterized the SARS-CoV-2-isolates by whole genome sequencing. Viral load measurements and sequence analysis were performed consecutively before and after bamlanivimab administration.FindingsAfter initial decrease of viral load, viral clearance was not achieved in five of six immunocompromised patients treated with bamlanivimab. Instead, viral replication increased again over the course of the following one to two weeks. In these five patients, the E484K substitution – known to confer immune escape – was detected at the time of viral rebound but not before bamlanivimab treatment.InterpretationTreatment of SARS-CoV-2 with bamlanivimab in immunocompromised patients results in the rapid development of immune escape variants in a significant proportion of cases. Given that the E484K mutation can hamper natural immunity, the effectiveness of vaccination as well as antibody-based therapies, these findings may have important implications not only for individual treatment decisions but may also pose a risk to general prevention and treatment strategies.FundingAll authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.

Anti-CD19 CAR T Cells That Secrete a Biparatopic Anti-CLEC12A Bridging Protein Have Potent Activity Against Highly Aggressive Acute Myeloid Leukemia In Vitro and In Vivo.

Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.

Angiopoietin-Like Protein 3 (ANGPTL3) Modulates Lipoprotein Metabolism and Dyslipidemia.

Dyslipidemia is characterized by increasing plasma levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides (TGs) and TG-rich lipoproteins (TGRLs) and is a major risk factor for the development of atherosclerotic cardiovascular disorders (ASCVDs). It is important to understand the metabolic mechanisms underlying dyslipidemia to develop effective strategies against ASCVDs. Angiopoietin-like 3 (ANGPTL3), a member of the angiopoietin-like protein family exclusively synthesized in the liver, has been demonstrated to be a critical regulator of lipoprotein metabolism to inhibit lipoprotein lipase (LPL) activity. Genetic, biochemical, and clinical studies in animals and humans have shown that loss of function, inactivation, or downregulated expression of ANGPTL3 is associated with an obvious reduction in plasma levels of TGs, LDL-C, and high-density lipoprotein-cholesterol (HDL-C), atherosclerotic lesions, and the risk of cardiovascular events. Therefore, ANGPTL3 is considered an alternative target for lipid-lowering therapy. Emerging studies have focused on ANGPTL3 inhibition via antisense oligonucleotides (ASOs) and monoclonal antibody-based therapies, which have been carried out in mouse or monkey models and in human clinical studies for the management of dyslipidemia and ASCVDs. This review will summarize the current literature on the important role of ANGPTL3 in controlling lipoprotein metabolism and dyslipidemia, with an emphasis on anti-ANGPTL3 therapies as a potential strategy for the treatment of dyslipidemia and ASCVDs.

Narrative review of pembrolizumab for the treatment of esophageal cancer: evidence and outlook.

ObjectiveBased on the current evidence, review the efficacy and safety profile of pembrolizumab, along with its shortcomings, in an effort to define future research directions.BackgroundThe survival outcome of esophageal cancer (EC) is poor, especially in patients with advanced stage. Palliative surgery, chemotherapy, radiotherapy and chemoradiotherapy have limited efficacy in prolonging the survival time. Currently, immunotherapies, including adoptive cell therapy-based, antibody-based, and vaccine-based therapies, are attracting considerable attention. The mechanism of immunotherapy lies in the modification of immune response and prevention of immune escape. Immunomodulatory agents can block the programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) pathway, thereby allowing lymphocytes to attack tumor cells. This class of drugs has the potential to treat a variety of tumors and may substantially improve overall survival (OS) in some patients. Multiple clinical trials have shown that pembrolizumab has good efficacy and safety, enhances the EC treatment paradigm, and has even become the first-line treatment of choice for patients with PD-L1-positive recurrent or metastatic EC.MethodsWe reviewed the results of clinical trials of pembrolizumab for EC and gastroesophageal cancer presented at Embase, PubMed, the American Society of Clinical Oncology (ASCO) annual meetings, and the Cochrane Central Register of Controlled Trials.ConclusionsPembrolizumab has good efficacy and tolerability profiles, and has emerged as a second-line option for the treatment of PD-L1-positive locally advanced or metastatic ESCC. Pembrolizumab has many promising applications, and further investigations into its mechanisms should be conducted.

Abstract 2225: Transposable elements are an abundant and pan-cancer source of shared tumor-specific antigens and membrane targets

Abstract Transposable elements (TEs) represent close to half of the genome and are generally disregarded in genomic studies due to their silencing in somatic tissues and difficulty in mapping to their repetitive sequences. Recent studies have revealed that epigenetic dysregulation in cancer can unlock the regulatory potential of transposable elements (TEs), and they can play an important role in cancer progression and oncogenesis. One important consequence of this phenomenon is the pervasive activation of TEs' intrinsic promoters, which leads to generation of thousands of unique transcripts. Many of these transcripts splice into downstream genes and lead to the generation of TE-gene chimeric transcripts. These transcripts can alter the main reading frame of the original transcript to generate unique isoforms of the target gene or generate novel out-of-frame peptides that could be therapeutic targets. In this study, we analyzed the transcriptomes of 11,092 samples from 33 TCGA cancer types and 675 cancer cell lines to comprehensively profile all TE-gene fusion transcripts. Using somatic tissues from FANTOM5 and GTEx, we filtered these transcripts for tumor-specificity and discovered 2,642 tumor-specific TE-gene transcripts that promiscuously occur in nearly all TCGA tumor samples. Computational prediction of reading frames of these transcripts identified 1,202 candidates with the potential to generate tumor-specific TE-derived antigens (TS-TEAs). We further analyzed tumor mass spectrometry data from breast adenocarcinoma and ovarian cancer and confirmed that unique peptide sequences from TS-TEAs could be detected. In addition, we performed HLA-pulldown mass spectrometry and confirmed that TS-TEAs are presented on the cell surface in cancer cell lines. Given that these antigens are highly shared within and across cancer types, we assessed their potential to generate universal antigen-based therapies. Optimal combinations of 5, 10, and 20 TS-TEAs could generate unique peptides that bind to patient-specific HLA alleles for 39.2%, 50.8%, and 60.8% of all TCGA tumors respectively. Lastly, we highlight the tumor-specific membrane proteins transcribed from TE-exapted promoters that can potentially expose novel epitopes on the extracellular surface of cancer cells. These can be valuable targets of CAR-T or alternative antibody-based therapies. In conclusion, we showcase the high prevalence of TE-derived promoter activation in cancer and suggest multiple avenues by which this phenomenon can be targeted therapeutically. Citation Format: Nakul M. Shah, Hyo Sik Jang, Ju Heon Maeng, Shin-Cheng Tzeng, Angela Wu, Changxu Fan, Noah L. Basri, Benjamin Katz, Daofeng Li, Xiaoyun Xing, Bradley S. Evans, Ting Wang. Transposable elements are an abundant and pan-cancer source of shared tumor-specific antigens and membrane targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2225.

Abstract 3038: Evaluation of ROR1-targeted antibody-drug conjugates against ROR1-expressing pediatric preclinical models - a report from the pediatric preclinical testing consortium (PPTC)

Abstract Introduction: ROR1 is an orphan-receptor tyrosine kinase-like surface antigen that is expressed primarily during development but is not expressed on normal adult tissues. ROR1 is expressed on multiple adult hematologic and solid cancers, making it a target for antibody-based therapies. UC-961 (cirmtuzumab), a humanized IgG1 monoclonal antibody, binds with high affinity to a specific extracellular epitope of human ROR1 and rapidly internalizes and traffics to lysosomes. VLS-101 is an ADC developed by conjugating UC-961 to a cleavable linker with an MMAE payload. VLS-211 was generated by conjugating UC-961 with a PNU payload. VLS-101 is in clinical development for adults with hematologic and solid cancers and has received FDA Fast Track and Orphan Drug designation for the treatment of patients with mantle cell lymphoma. To better define the utility of ROR1 as an immuno-oncology target for pediatric cancers, we evaluated VLS-101 and VLS-211 against pediatric preclinical models selected based on ROR1 expression. Methods: Models were selected based on RNA-seq transcript levels for ROR1 with subsequent confirmation by flow cytometry for acute lymphoblastic leukemia (ALL) models. For the ALL models, UC-961 (5 mg/kg), VLS-101 (2.5 and 5.0 mg/kg), and VLS-211 (0.25 and 0.5 mg/kg) were each administered weekly x 3. For the Ewing sarcoma (EWS) models, the dose of VLS-211 was 0.5 and 1 mg/kg. Four models were treated using a twice-weekly or every 4-day treatment schedule while 2 models were treated weekly. Results: Elevated ROR1 gene expression among PPTC models was most notable among the ALL and EWS panels. Leukemia cell surface expression of ROR1 correlated with transcript levels. ALL models with elevated ROR1 expression [TCF3-HLF (1), TCF3-PBX1 (2), ETV6-RUNX1 (2), and BCR-ABL1 (1)] as well as a non-expressing model were tested against the ROR1 ADCs. Activity of each ADC was ROR1 expression-level dependent, and for VLS-211 dose-dependency was noted. At the highest doses of each ADC studied, 2 of 6 (VLS-101) and 3 of 6 (VLS-211) ROR1-expressing models showed objective responses. For most models, median time to event was longer for VLS-211 versus VLS-101. Five EWS models were studied, and expression-level dependent activity was observed for both ADCs. VLS-101 induced tumor regressions in 2 of 5 models, while VLS211 induced regressions in 3 of 5 models. Time to event was generally longer for VLS-211 compared to VLS-101. No objective responses were observed to UC-961 for the ALL and EWS models. Conclusions: VLS-101 and VLS-211 showed expression-level dependent activity against ALL and EWS models. These results support ROR1 as a relevant immuno-oncology target for the subset of B-ALL and Ewing sarcoma cases with elevated ROR1 expression. Further research is required to identify the optimal payload for ROR1 ADCs for use against pediatric cancers. Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Brian J. Lannutti, Katti A. Jessen, Eric J. Earley, Stephen W. Erickson, Malcolm A. Smith, Raushan Kurmasheva, Peter J. Houghton. Evaluation of ROR1-targeted antibody-drug conjugates against ROR1-expressing pediatric preclinical models - a report from the pediatric preclinical testing consortium (PPTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3038.

The Evolving Impact on Survival Outcomes in Multiple Myeloma with Greater Access to Novel Treatment Regimens: Experience in a Real-World Cohort

The Evolving Impact on Survival Outcomes in Multiple Myeloma with Greater Access to Novel Treatment Regimens: Experience in a Real-World Cohort

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Bispecific Anti-CD123 x Anti-CD3 Adaptir™ Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro

Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML. DisclosuresGottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

Altered Expression of CCR10 Indicates Malignant Transformation of Bone Marrow Plasma Cells

Introduction. Phenotypic analysis of tumor cells by flow cytometry is now essential for diagnosis and treatment of hematological malignancies, including multiple myeloma (MM). Surface molecules such as CD19, CD56, CD27 and CD45 have been used to distinguish normal bone marrow (BM) plasma cells from malignant plasma cells (MM cells). Molecules that are relatively specific to plasma cells (CD38 and SLAMF7) are considered targets for antibody-based therapies in MM. Among the vast numbers of surface molecules, chemokine receptors have been attracting much attention as the regulators of migration and tissue localization of plasma cells and MM cells. CXCR4 (C-X-C chemokine receptor type 4) has been reported to guide the movement of plasma cells and myeloma cells to the BM, where its ligand CXCL12 (C-X-C motif chemokine 12) is abundantly produced. CCR10 (C-C motif chemokine receptor 10) is another chemokine receptor, whose ligand is CCL27 (C-C motif chemokine ligand 27) and CCL28, also known to be involved in cell homing and migration. CCR10 has been reported to be highly expressed on mucosal derived plasma cells, however its expression in MM cells and its role within the BM microenvironment has not been well studied. In this study, we analyzed the expression of CCR10 on BM lymphocytes from patients of plasma cell disorders in order to understand its significance in the BM microenvironment.Materials and methods. We analyzed a public available gene expression dataset (GSE 22886) in order to study CCR10 gene expression in human plasma cells. To examine CCR10 gene expression in myeloma cells, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). Multi-color flow cytometry was performed to study CCR10 expression on bone marrow lymphocytes. BM plasma cells were determined as CD38++ CD138+ or CD38++ SLAMF7+. Monoclonal plasma cells (myeloma clones) were determined as CD19- CD56- or CD19- CD56+ cells and polyclonal plasma cells (normal plasma cells) as CD19+ CD56- cells. CCR10 expression of BM plasma cells, B cells, CD4 T cells, CD8 T cells and NK cells of MGUS, newly diagnosed MM, relapsed-refractory MM patients were analyzed.Results. Gene expression dataset analysis showed that, among the normal leukocyte subsets, plasma cells had the highest CCR10 gene expression compared to other leukocytes (p<0.001). Myeloma cell lines showed the highest expression of CCR10 gene expression among all the other cancer cell lines, indicating the specificity of CCR10 expression in MM cells. Multi-color flow cytometry showed significantly high expression of CCR10 on BM plasma cells compared to other lymphocytes in the bone marrow of MGUS patients (p<0.0001). Within the MGUS BM plasma cells, monoclonal plasma cells had significantly lower CCR10 expression compared to polyclonal plasma cells (p<0.05). Additionally, monoclonal plasma cells from relapsed-refractory MM patients had significantly lower CCR10 expression compared to those from MGUS and newly diagnosed MM patients. These data indicate a role of CCR10 decrease in malignant plasma cells leading to MM progression. We further analyzed other surface molecules that may be related to CCR10 on monoclonal plasma cells. CCR10 expression did not correlate with CD56 expression, while CD45- monoclonal plasma cells tend to have lower CCR10 expression compared to the CD45+ counterparts in newly diagnosed MM patients (p<0.001).Conclusions. Gene expression dataset analysis and multi-color flow cytometry have showed that CCR10 is highly expressed in normal plasma cells. However, we show here for the first time that plasma cells start to decrease CCR10 expression at malignant transformation and during disease progression. Since, CCL27, the ligand of CCR10, is known to induce chemotaxis to plasma cells, loss of CCR10 may lead to migration or metastasis of MM cells to different sites. Further research is now on-going to elucidate the significance and mechanism of altered CCR10 expression in MM cells. This study may lead to a better understanding of MM biology and to the development of a novel strategy to target malignant plasma cells. DisclosuresNo relevant conflicts of interest to declare.

Harnessing the power of antibodies to fight bone metastasis.

Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.