SGN-B6A: A New Vedotin Antibody-Drug Conjugate Directed to Integrin Beta-6 for Multiple Carcinoma Indications.

Integrin beta-6, a component of the heterodimeric adhesion receptor alpha-v/beta-6, is overexpressed in numerous solid tumors. Its expression has been shown by multiple investigators to be a negative prognostic indicator in diverse cancers including colorectal, non-small cell lung, gastric, and cervical. We developed SGN-B6A as an antibody-drug conjugate (ADC) directed to integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE) to cancer cells. The antibody component of SGN-B6A is specific for integrin beta-6 and does not bind other alpha-v family members. In preclinical studies, this ADC has demonstrated activity in vivo in models derived from non-small cell lung, pancreatic, pharyngeal, and bladder carcinomas spanning a range of antigen expression levels. In nonclinical toxicology studies in cynomolgus monkeys, doses of up to 5 mg/kg weekly for four doses or 6 mg/kg every 3 weeks for two doses were tolerated. Hematologic toxicities typical of MMAE ADCs were dose limiting, and no significant target-mediated toxicity was observed. A phase I first-in-human study is in progress to evaluate the safety and antitumor activity of SGN-B6A in a variety of solid tumors known to express integrin beta-6 (NCT04389632).

Development of Novel Antibody-Camptothecin Conjugates.

We have developed a highly active and well-tolerated camptothecin (CPT) drug-linker designed for antibody-mediated drug delivery in which the lead molecule consists of a 7-aminomethyl-10,11-methylenedioxy CPT (CPT1) derivative payload attached to a novel hydrophilic protease-cleavable valine-lysine-glycine tripeptide linker. A defined polyethylene glycol stretcher was included to improve the properties of the drug-linker, facilitating high antibody-drug conjugate (ADC) drug loading, while reducing the propensity for aggregation. A CPT1 ADC with 8 drug-linkers/mAb displayed a pharmacokinetic profile coincident with parental unconjugated antibody and had high serum stability. The ADCs were broadly active against cancer cells in vitro and in mouse xenograft models, giving tumor regressions and complete responses at low (≤3 mg/kg, single administration) doses. Pronounced activities were obtained in both solid and hematologic tumor models and in models of bystander killing activity and multidrug resistance. Payload release studies demonstrated that two CPTs, CPT1 and the corresponding glycine analog (CPT2), were released from a cAC10 ADC by tumor cells. An ADC containing this drug-linker was well tolerated in rats at 60 mg/kg, given weekly four times. Thus, ADCs comprised of this valine-lysine-glycine linker with CPT drug payloads have promise in targeted drug delivery.

A coiled-coil masking domain for selective activation of therapeutic antibodies.

The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.

Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies.

Patients with relapsed/refractory B-cell malignancies such as non-Hodgkin lymphoma (B-NHL) or acute lymphoblastic leukemia have a poor prognosis. Despite measurable clinical activity with new targeted therapies, many patients do not achieve a complete or durable response suggesting an opportunity to improve upon existing therapies. Here we describe SGN-CD19B, a pyrrolobenzodiazepine (PBD)-based anti-CD19 antibody drug conjugate (ADC) being investigated for treatment of B-cell malignancies, which has improved potency compared with other ADCs. CD19-expressing tumor cells rapidly internalize SGN-CD19B, and the released PBD drug induces DNA damage, resulting in G2/M cell cycle arrest and cell death. SGN-CD19B demonstrated activity against a broad panel of malignant B-cell lines and induced durable regressions in mice bearing xenografts derived from these B-cell malignancies. A single dose of SGN-CD19B induced durable regressions at 300 µg/kg (3 µg/kg drug equivalents); combination with rituximab decreased the curative dose to 100 µg/kg (1 µg/kg drug equivalents). These doses are significantly lower than the level of drug required with other ADC payloads. In cynomolgus monkeys, SGN-CD19B effectively depleted CD20+ B lymphocytes in peripheral blood and lymphoid tissues confirming that SGN-CD19B is pharmacodynamically active at well-tolerated doses. In summary, preclinical studies show SGN-CD19B is a highly active ADC, which releases a DNA cross-linking agent rather than a microtubule inhibitor. The distinct mechanism of action, broad potency, and potential to combine with rituximab suggest that SGN-CD19B may offer unique clinical opportunities in B-cell malignancies. A phase 1 clinical trial is in progress to investigate the therapeutic potential of SGN-CD19B in relapsed/refractory B-NHL. This trial was registered at www.clinicaltrials.gov as #NCT02702141.

SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

Targeting of BAFF and APRIL for autoimmunity and oncology.

BLyS and APRIL are tumor necrosis factor superfamily members shown to be important for B-cell development, maturation and survival. Recent data also indicate that these cytokines regulate the survival and maintenance of malignant B-cells in cancer patients. The key role of BlyS/APRIL in the immune system and their potential role in cancers have attracted the attention of basic scientistis and biotechnology companies alike. As a result, the pathways regulated by BLYS and APRIL have been quickly elevated as attractive targets for antibody-based and non-antibody based therapeutics. Exploitation of these pathways has not only given us enormous insights into the basic biology of the APRIL/BLyS system but it has also identified potential clinical candidates for cancer and autoimmunity. As such, multiple biotechnology companies are currently testing various therapeutic candidates in the clinic. This chapter will review BLyS and APRIL functions and discuss alternative therapeutic approaches to target BLyS and APRIL pathways for human malignancies and autoimmunity.

Antibody targeting of B-cell maturation antigen on malignant plasma cells.

B-cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, we developed antibodies with ligand blocking activity that could promote cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. We show that SG1, an inhibitory BCMA antibody, blocks APRIL-dependent activation of nuclear factor-kappaB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance FcgammaRIIIA binding. The Fc mutations increased the antibody-dependent cell-mediated cytotoxicity potency of BCMA antibodies against MM lines by approximately 100-fold with a > or = 2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody-drug conjugate displayed IC(50) values of < or = 130 pmol/L for three different MM lines. Hence, BCMA antibodies show cytotoxic activity both as naked IgG and as drug conjugates and warrant further evaluation as therapeutic candidates for plasma cell malignancies.

Lymphocyte activation antigen CD70 expressed by renal cell carcinoma is a potential therapeutic target for anti-CD70 antibody-drug conjugates.

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

Globular domains 4/5 of the laminin alpha3 chain mediate deposition of precursor laminin 5.

In epidermal wounds, precursor laminin 5 (alpha3beta3gamma2) is deposited in the provisional basement membrane (PBM) before other BM components. Precursor laminin 5 contains G4/5 globular domains at the carboxyl terminus of the alpha3 chain. Here, the function of G4/5 was evaluated in deposition of laminin 5. Soluble laminin 5, secreted by keratinocytes in culture, is cleaved by an endogenous protease releasing G4/5. Thrombin, a serum protease, cleaves G4/5 indistinguishably from endogenous protease. Soluble human precursor laminin 5, but not cleaved laminin 5, was bound and deposited by mouse keratinocytes null for mouse alpha3 chain (alpha3-/- MKs). The deposition rescued adhesion and spreading and survival. In a model for PBM assembly, precursor laminin 5 was deposited along fibronectin fibrils at the junction between co-cultures of keratinocytes and fibroblasts. In both models, the deposition of precursor laminin 5 was inhibited by removal of G4/5 with thrombin. To confirm that G4/5 participates in deposition, the human LAMA3A gene was modified to produce alpha3 chains either without or with G4/5 that cannot be cleaved. Both precleaved and noncleavable alpha3 isoforms were expressed in alpha3-/- MKs, where they deposited sufficiently to rescue adhesion via integrins alpha3beta1 and alpha6beta4. Despite this similarity, noncleavable laminin 5 was at least threefold more efficiently deposited than precleaved isoform. We conclude that the G4/5 domain in the alpha3 chain facilitates deposition of precursor laminin 5 into the PBM in epidermal wounds.