Andrew Polson

Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkins lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkins lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Phosphorylated by Cyclin-Dependent Kinases

ABSTRACT The K8 locus in Kaposis sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.

FcRL5 as a Target of Antibody–Drug Conjugates for the Treatment of Multiple Myeloma

Fc receptor-like 5 (FcRL5/FcRH5/IRTA2/CD307) is a surface protein expressed selectively on B cells and plasma cells. We found that FcRL5 was expressed at elevated levels on the surface of plasma cells from the bone marrow of patients diagnosed with multiple myeloma. This prevalence in multiple myeloma and narrow pattern of normal expression indicate that FcRL5 could be a target for antibody-based therapies for multiple myeloma, particularly antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, where limited expression on normal tissues is a key component to their safety. We found that FcRL5 is internalized upon antibody binding, indicating that ADCs to FcRL5 could be effective. Indeed, we found that FcRL5 ADCs were efficacious in vitro and in vivo but the unconjugated antibody was not. The two most effective consisted of our anti-FcRL5 antibody conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (anti-FcRL5-MC-vcPAB-MMAE) or conjugated via lysines to the maytansinoid DM4 through a disulfide linker (anti-FcRL5-SPDB-DM4). These two ADCs were highly effective in vivo in combination with bortezomib or lenalidomide, drugs in use for the treatment of multiple myeloma. These data show that the FcRL5 ADCs described herein show promise as an effective treatment for multiple myeloma. Mol Cancer Ther; 11(10); 2222–32. ©2012 AACR.

Investigational antibody-drug conjugates for hematological malignancies

Importance of the field: Antibody-drug conjugates (ADCs) consist of potent cytotoxic drugs linked to antibodies via chemical linkers. ADCs facilitate the specific targeting of drugs to neoplastic cells. This technology is showing efficacy with manageable toxicity for the treatment of hematological malignancies. Areas covered in this review: ADCs for the treatment of hematological malignancies are in pre-clinical and early clinical trials. This review describes these ADCs in detail and explores the challenges of optimizing the use of this technology. What the reader will gain: The reader should understand that, although ADCs are conceptually simple, the application of this idea to practice has not been straightforward, and the challenges of developing ADCs include identifying targets with appropriate expression profiles and biology, developing successful linker chemistries, and the selection of a potent cytotoxic drug. Take home message: Hematological malignancies are particularly suited to the development of ADC therapeutics as their surface proteins are well characterized, and the consequences of expression of the target in the normal tissue like the bone marrow results in manageable toxicities since, in many cases, the normal tissue can regenerate. While this technology is complex, the ADCs for hematological malignancies currently in clinical use show great promise.

Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Non-Cleavable Drug-Linkers.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Stabilizing a Tubulysin Antibody–Drug Conjugate To Enable Activity Against Multidrug-Resistant Tumors

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro. However, we observed metabolism of a critical acetate ester of the drug in vivo, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

ABSTRACT For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.

Abstract 3628: T cell-dependent bispecific antibody anti-CD79b/CD3 as a potential therapy for B-cell malignancies

T-cell recruiting bispecific antibodies and antibody fragments have been used to harness the cytotoxic potential of T cells for cancer treatment. We have adopted a knobs-into-holes antibody format and produced T-cell dependent bispecific antibodies (TDB), as full length, humanized IgG1 antibodies with natural antibody architecture, which allow one arm to target various B cell antigens while the other arm recruits T cells by binding to the CD3e subunit of the T-cell receptor. One B cell antigen targeted is CD79b, a component of the B cell receptor complex, which has been clinically validated by an anti-CD79b antibody-drug conjugate (ADC) as a safe and effective therapeutic target for B cell malignancies (Leukemia 2015 Pfeifer et.al). In the present work, we show that anti-CD79b/CD3 TDB is active against lymphoma cells with a wide range of CD79b antigen levels in vitro. In addition, anti-CD79b/CD3 TDB appears to be insensitive to the status of cellular signaling pathways in lymphoma cells as it is active against cell lines that are resistant to the BTK inhibitor ibrutinib or an anti-CD79b ADC with a cleavable tubulin inhibitor as the payload. In vivo, anti-CD79b/CD3 TDB administration inhibited tumor growth in B-cell lymphoma xenograft models and resulted in potent B-cell depletion in the blood and spleens in a humanized murine model. To assess the safety of targeting CD79b with a T-cell recruiting bispecific antibody in non-human primates, a surrogate anti-cynoCD79b/CD3 TDB with comparable in vitro potency was produced with a target arm that recognizes cynomolgus monkey CD79b and the same anti-CD3 arm. In a single dose pharmacokinetic/pharmacodynamics/safety study, anti-cynoCD79b/CD3 TDB administration at 1mg/kg resulted in potent B cell depletion, as well as T cell activation and proliferation, which was assessed by flow cytometry on blood and lymphoid tissue and immunohistochemistry on lymphoid tissue. The pharmacokinetic properties of anti-cynoCD79b/CD3 TDB resembled that of IgG antibodies; though with a faster clearance likely due to CD79b antigen internalization and enhanced binding to CD3 when compared to previously described anti-CD20/CD3 TDB. Transient cytokine release was observed as elevated levels of IL-2, IL-6, IFN-gamma and TNF-alpha were detected within 24 hours following administration. Anti-cynoCD79b/CD3 TDB was well-tolerated in the majority of dosed animals without toxicologically significant findings. Collectively, these preclinical data suggest anti-CD79b/CD3 TDB can be a potential therapeutic agent in B cell malignancies. Citation Format: Peiyin Wang, Maria Hristopoulos, Robyn Clark, Yvonne Chen, Diego Ellerman, Mary Mathieu, Christoph Spiess, Jessica Li, Cecile Chalouni, Siddharth Sukumaran, Eric Stefanich, Jeffrey Wallin, Robert Li, Tanja Zabka, Klara Totpal, Mark Dennis, Allen Ebens, Stephen Gould, Andrew Polson, Liping Laura Sun. T cell-dependent bispecific antibody anti-CD79b/CD3 as a potential therapy for B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2017-3628