Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Antibody Drug Conjugates for Cancer Therapy.

Antibody drug conjugates (ADCs) constitute a family of cancer therapeutics designed to preferentially direct a cytotoxic drug to cells expressing a cell-surface antigen recognized by an antibody. The antibody and drug are linked through chemistries that enable release of the cytotoxic drug or drug adduct upon internalization and digestion of the ADC by the cell. Over 40 distinct ADCs, targeting an array of antigens and utilizing a variety of drugs and linkers, are undergoing clinical evaluation. This review primarily covers ADCs that have advanced to clinical investigation with a particular emphasis on how the individual targets, linker chemistries, and appended drugs influence their behavior.

High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Drugging Wnt signalling in cancer.

Aberrant regulation of the Wnt signalling pathway has emerged as a prevalent theme in cancer biology. This chapter summarizes the research that provides a proof of concept for inhibiting Wnt signalling in cancer, the potential means by which this could be achieved, and some recent advances towards this goal. A brief discussion of molecular diagnostics and possible safety concerns is also provided.

An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.

Reversible modification of adenomatous polyposis coli (APC) with K63-linked polyubiquitin regulates the assembly and activity of the ß-catenin destruction complex.

The adenomatous polyposis coli (APC) tumor suppressor forms a complex with Axin and GSK3ß to promote the phosphorylation and degradation of ß-catenin, a key co-activator of Wnt-induced transcription. Here, we establish that APC is modified predominantly with K63-linked ubiquitin chains when it is bound to Axin in unstimulated HEK293 cells. Wnt3a stimulation induced a time-dependent loss of K63-polyubiquitin adducts from APC, an effect synchronous with the dissociation of Axin from APC and the stabilization of cytosolic ß-catenin. RNAi-mediated depletion of Axin or ß-catenin, which negated the association between APC and Axin, resulted in the absence of K63-adducts on APC. Overexpression of wild-type and phosphodegron-mutant ß-catenin, combined with analysis of thirteen human cancer cell lines that harbor oncogenic mutations in APC, Axin, or ß-catenin, support the hypothesis that a fully assembled APC-Axin-GSK3ß-phospho-ß-catenin complex is necessary for the K63-polyubiquitylation of APC. Intriguingly, the degree of this modification on APC appears to correlate inversely with the levels of ß-catenin in cells. Together, our results indicate that K63-linked polyubiquitin adducts on APC regulate the assembly and/or efficiency of the ß-catenin destruction complex.

The melanosomal protein PMEL17 as a target for antibody drug conjugate therapy in melanoma.

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

The many ways of Wnt in cancer.

More than 20 years ago, the oncogenicity of a Wnt ligand was revealed in a series of experiments originating with random proviral integration in mice. The significance of Wnt signaling in human cancer has since been buttressed by the identification of mutations in genes coding for the Wnt pathway components Axin, APC, and beta-catenin. This review summarizes the reported genetic defects in the Wnt pathway, with an emphasis on their functional contribution to human tumor progression.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.

Preclinical evaluation of carcinoembryonic cell adhesion molecule (CEACAM) 6 as potential therapy target for pancreatic adenocarcinoma.

Despite the availability of new targeted therapies, ductal pancreatic adenocarcinoma continues to carry a poor prognosis. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM)6 has been reported as a potential biomarker and therapy target for this malignancy. We have evaluated CEACAM6 as a potential therapy target, using an antibody-drug conjugate (ADC). Expression of CEACAM6 in pancreatic adenocarcinomas was determined using immunohistochemistry on tissue microarrays. The expression pattern in granulocytes and granulocytic precursors was measured by flow cytometry. Murine xenograft and non-human primate models served to evaluate efficacy and safety, respectively. Robust expression of CEACAM6 was found in > 90% of invasive pancreatic adenocarcinomas as well as in intraepithelial neoplastic lesions. In the granulocytic lineage, CEACAM6 was expressed at all stages of granulocytic maturation except for the early lineage-committed precursor cell. The anti-CEACAM6 ADC showed efficacy against established CEACAM6-expressing tumours. In non-human primates, antigen-dependent toxicity of the ADC consisted of dose-dependent and reversible depletion of granulocytes and their precursors. This was associated with preferential and rapid localization of the antibody in bone marrow, as determined by sequential in vivo PET imaging of the radiolabelled anti-CEACAM6. Localization of the radiolabelled tracer could be attenuated by predosing with unlabelled antibody confirming specific accumulation in this compartment. Based on the expression pattern in normal and malignant pancreatic tissues, efficacy against established tumours and limited and reversible bone marrow toxicity, we propose that CEACAM6 should be considered for an ADC-based therapy approach against pancreatic adenocarcinomas and possibly other CEACAM6-positive neoplasms.

Identification and immunotherapeutic targeting of antigens induced by chemotherapy.

Cancer cells differ from normal cells in their response to chemotherapy. We exploited this dissimilarity by identifying and targeting tumor-specific, cell-surface proteins whose expression is induced by the chemotherapeutic irinotecan (CPT-11; Camptosar). A cytotoxin-armed antibody reactive with one of these drug-induced surface proteins, the LY6D/E48 antigen, originally identified as the target of a monoclonal antibody reactive with squamous cell carcinomas, caused complete regression of colorectal tumor xenografts in mice treated with CPT-11, whereas either agent alone was less effective. These results suggest that a positive therapeutic index may be generated for other drug combinations by immunotherapeutic targeting of chemotherapy-induced antigens.

The soluble wnt receptor Frizzled8CRD-hFc inhibits the growth of teratocarcinomas in vivo.

Wnt signaling is important for normal cell proliferation and differentiation, and mutations in pathway components are associated with human cancers. Recent studies suggest that altered wnt ligand/receptor interactions might also contribute to human tumorigenesis. Therefore, agents that antagonize wnt signaling at the extracellular level would be attractive therapeutics for these cancers. We have generated a soluble wnt receptor comprising the Frizzled8 cysteine-rich domain (CRD) fused to the human Fc domain (F8CRDhFc) that exhibits favorable pharmacologic properties in vivo. Potent antitumor efficacy was shown using the mouse mammary tumor virus-Wnt1 tumor model under dosing conditions that did not produce detectable toxicity in regenerating tissue compartments. In vitro, F8CRDhFc inhibited autocrine wnt signaling in the teratoma cell lines PA-1, NTera-2, Tera-2, and NCCIT. In vivo, systemic administration of F8CRDhFc significantly retarded the growth of tumor xenografts derived from two of these cell lines, PA-1 and NTera-2. Pharmacodynamic markers of wnt signaling, identified by gene expression analysis of cultured teratoma cells, were also modulated in the tumor xenografts following treatment with F8CRDhFc. Additionally, these markers could be used as indicators of treatment efficacy and might also be useful in identifying patients that would benefit from the therapeutic agent. This is the first report showing the efficacy of a soluble wnt receptor as an antitumor agent and suggests that further development of wnt antagonists will have utility in treating human cancer.

Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models.

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences ("mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety.

An Anti-GDNF Family Receptor Alpha 1 (GFRA1) Antibody-Drug Conjugate for the Treatment of Hormone Receptor-Positive Breast Cancer.

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.

Casein kinase 1: a Wnt'er of disconnect.

Phosphorylation of beta-catenin, a central downstream component of the Wnt pathway, by glycogen synthase kinase 3 is essential for its targeted degradation by the proteosome. New studies show that casein kinase 1 primes beta-catenin for subsequent phophorylation by glycogen synthase kinase 3.

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.

EphB2 is a prognostic factor in colorectal cancer.

A receptor tyrosine kinase for ephrin ligands, EphB2 is expressed in colorectal cancer and has been proposed as a target for immunoconjugate therapy. The aim of this study was to perform a detailed histologic analysis of EphB2 expression in normal and neoplastic colorectal tissues. In addition, we sought to evaluate EphB2 expression as a prognostic factor in colorectal cancer. Expression of EphB2 was examined in normal colon (n = 28), colorectal cell lines (n = 20), colorectal adenomas (n = 148), primary cancers (n = 28), and metastases (n = 39) using immunohistochemistry. In addition, a series of primary cancers and matched normal (n = 342) with outcome data were profiled in tissue microarrays. The intensity of EphB2 expression was assessed in the entire series by immunohistochemistry, and in a subset by in situ hybridization. Overall survival and recurrence-free survival were correlated with EphB2 protein expression in retrospective subset analyses. Epithelial EphB2 expression was shown at all stages of colorectal tumorigenesis, including the base of all normal crypts, 77% of adenomas, 82% of primary cancers, and 64% of metastases. Although homogeneous expression was observed in adenomas, the pattern of staining was focal (mean 25%) in most malignant lesions. Patients whose tumor stained 2+ for EphB2 expression (versus 0/1+) exhibited significantly prolonged overall survival: mean duration of survival, 2,514 versus 1,044 days; hazard ratio, 0.45; 95% confidence interval, 0.18 to 0.95 (P = 0.035). In summary, EphB2 is expressed in normal crypts, colorectal adenomas, primary cancers, and metastases. High levels of EphB2 expression are associated with a longer mean duration of survival in colorectal cancer.

Arming antibodies for cancer therapy.

Specifying the delivery of a highly toxic agent to cancers while sparing normal tissue is an attractive prospect for the treatment of cancer. Cell surface antigens with highly restricted expression patterns have been identified and the means by which cytotoxic agents are appended to antibodies has been greatly refined. Myriad formulations involving radionuclides, bacterial toxins and small-molecule drugs linked to antibodies through peptides, protein fusions and chelating agents are in preclinical and clinical evaluation, and a few have been approved as human medicines. Recent advances hold promise for improving the therapeutic index of these anti-cancer drugs.

Hath1, down-regulated in colon adenocarcinomas, inhibits proliferation and tumorigenesis of colon cancer cells.

A striking feature of colon tumors is the significant reduction of goblet cells. Although targeted deletion of Math1 in mice leads to a loss of intestinal secretory cells, including goblet cells, the role of Hath1 in colon tumorigenesis remains unknown. Here we report that Hath1, the human ortholog of Math1, was dramatically down-regulated in colon tumor samples and colon cancer cell lines. Overexpression of Hath1 in HT29, an aggressive colon cancer cell line, resulted in a significant inhibition on cell proliferation, anchorage-independent growth in soft agar and, more importantly, growth of human colon cancer cell xenografts in athymic nude mice. Such inhibition was accompanied by altered expression of a goblet cell differentiation marker, MUC2, and cell cycle regulators cyclin D1 and p27kip1. Hath1 expression also was up-regulated on inhibition of the Wnt pathway, which has been well implicated in colon tumorigenesis. Hence, this study suggests that Hath1 may be a novel factor downstream of the Wnt pathway capable of suppressing anchorage-independent growth of colon cancer cell lines. More importantly, this study is the first to establish a link between down-regulation of Hath1 expression and colon tumorigenesis.