Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody-Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation.

Antibody-drug conjugates have elicited great interest recently as targeted chemotherapies for cancer. Recent preclinical and clinical data have continued to raise questions about optimizing the design of these complex therapeutics. Biochemical methods for site-specific antibody conjugation have been a design feature of recent clinical ADCs, and preclinical reports suggest that site-specifically conjugated ADCs generically offer improved therapeutic indices (i.e., the fold difference between efficacious and maximum tolerated doses). Here we present the results of a systematic preclinical comparison of ADCs embodying the DNA-alkylating linker-payload DGN549 generated with both heterogeneous lysine-directed and site-specific cysteine-directed conjugation chemistries. Importantly, the catabolites generated by each ADC are the same regardless of the conjugation format. In two different model systems evaluated, the site-specific ADC showed a therapeutic index benefit. However, the therapeutic index benefit is different in each case: both show evidence of improved tolerability, though with different magnitudes, and in one case significant efficacy improvement is also observed. These results support our contention that conjugation chemistry of ADCs is best evaluated in the context of a particular antibody, target, and linker-payload, and ideally across multiple disease models.

A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells.

The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.

IMGN779, a Novel CD33-Targeting Antibody-Drug Conjugate with DNA-Alkylating Activity, Exhibits Potent Antitumor Activity in Models of AML.

The myeloid differentiation antigen CD33 has long been exploited as a target for antibody-based therapeutic approaches in acute myeloid leukemia (AML). Validation of this strategy was provided with the approval of the CD33-targeting antibody-drug conjugate (ADC) gemtuzumab ozogamicin in 2000; the clinical utility of this agent, however, has been hampered by safety concerns. Thus, the full potential of CD33-directed therapy in AML remains to be realized, and considerable interest exists in the design and development of more effective ADCs that confer high therapeutic indices and favorable tolerability profiles. Here, we describe the preclinical characterization of a novel CD33-targeting ADC, IMGN779, which utilizes a unique DNA-alkylating payload to achieve potent antitumor effects with good tolerability. The payload, DGN462, is prototypical of a novel class of purpose-created indolinobenzodiazeprine pseudodimers, termed IGNs. With low picomolar potency, IMGN779 reduced viability in a panel of AML cell lines in vitro Mechanistically, the cytotoxic activity of IMGN779 involved DNA damage, cell-cycle arrest, and apoptosis consistent with the mode of action of DGN462. Moreover, IMGN779 was highly active against patient-derived AML cells, including those with adverse molecular abnormalities, and sensitivity correlated to CD33 expression levels. In vivo, IMGN779 displayed robust antitumor efficacy in multiple AML xenograft and disseminated disease models, as evidenced by durable tumor regressions and prolonged survival. Taken together, these findings identify IMGN779 as a promising new candidate for evaluation as a novel therapeutic in AML. Mol Cancer Ther; 17(6); 1271-9. ©2018 AACR.

Dual Inhibition of IGF-1R and ErbB3 Enhances the Activity of Gemcitabine and Nab-Paclitaxel in Preclinical Models of Pancreatic Cancer.

Purpose: Insulin-like growth factor receptor 1 (IGF-1R) is critically involved in pancreatic cancer pathophysiology, promoting cancer cell survival and therapeutic resistance. Assessment of IGF-1R inhibitors in combination with standard-of-care chemotherapy, however, failed to demonstrate significant clinical benefit. The aim of this work is to unravel mechanisms of resistance to IGF-1R inhibition in pancreatic cancer and develop novel strategies to improve the activity of standard-of-care therapies.Experimental Design: Growth factor screening in pancreatic cancer cell lines was performed to identify activators of prosurvival PI3K/AKT signaling. The prevalence of activating growth factors and their receptors was assessed in pancreatic cancer patient samples. Effects of a bispecific IGF-1R and ErbB3 targeting antibody on receptor expression, signaling, cancer cell viability and apoptosis, spheroid growth, and in vivo chemotherapy activity in pancreatic cancer xenograft models were determined.Results: Growth factor screening in pancreatic cancer cells revealed insulin-like growth factor 1 (IGF-1) and heregulin (HRG) as the most potent AKT activators. Both growth factors reduced pancreatic cancer cell sensitivity to gemcitabine or paclitaxel in spheroid growth assays. Istiratumab (MM-141), a novel bispecific antibody that blocks IGF-1R and ErbB3, restored the activity of paclitaxel and gemcitabine in the presence of IGF-1 and HRG in vitro Dual IGF-1R/ErbB3 blocking enhanced chemosensitivity through inhibition of AKT phosphorylation and promotion of IGF-1R and ErbB3 degradation. Addition of istiratumab to gemcitabine and nab-paclitaxel improved chemotherapy activity in vivoConclusions: Our findings suggest a critical role for the HRG/ErbB3 axis and support the clinical exploration of dual IGF-1R/ErbB3 blocking in pancreatic cancer. Clin Cancer Res; 24(12); 2873-85. ©2018 AACR.

MM-141, an IGF-IR- and ErbB3-directed bispecific antibody, overcomes network adaptations that limit activity of IGF-IR inhibitors.

Although inhibition of the insulin-like growth factor (IGF) signaling pathway was expected to eliminate a key resistance mechanism for EGF receptor (EGFR)-driven cancers, the effectiveness of IGF-I receptor (IGF-IR) inhibitors in clinical trials has been limited. A multiplicity of survival mechanisms are available to cancer cells. Both IGF-IR and the ErbB3 receptor activate the PI3K/AKT/mTOR axis, but ErbB3 has only recently been pursued as a therapeutic target. We show that coactivation of the ErbB3 pathway is prevalent in a majority of cell lines responsive to IGF ligands and antagonizes IGF-IR-mediated growth inhibition. Blockade of the redundant IGF-IR and ErbB3 survival pathways and downstream resistance mechanisms was achieved with MM-141, a tetravalent bispecific antibody antagonist of IGF-IR and ErbB3. MM-141 potency was superior to monospecific and combination antibody therapies and was insensitive to variation in the ratio of IGF-IR and ErbB3 receptors. MM-141 enhanced the biologic impact of receptor inhibition in vivo as a monotherapy and in combination with the mTOR inhibitor everolimus, gemcitabine, or docetaxel, through blockade of IGF-IR and ErbB3 signaling and prevention of PI3K/AKT/mTOR network adaptation.

Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3.

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

PT-100, a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism.

The amino boronic dipeptide, PT-100 (Val-boro-Pro), a dipeptidyl peptidase (DPP) inhibitor, has been shown to up-regulate gene expression of certain cytokines in hematopoietic tissue via a high-affinity interaction, which appears to involve fibroblast activation protein. Because fibroblast activation protein is also expressed in stroma of lymphoid tissue and tumors, the effect of PT-100 on tumor growth was studied in mice in vivo. PT-100 has no direct cytotoxic effect on tumors in vitro. Oral administration of PT-100 to mice slowed growth of syngeneic tumors derived from fibrosarcoma, lymphoma, melanoma, and mastocytoma cell lines. In WEHI 164 fibrosarcoma and EL4 and A20/2J lymphoma models, PT-100 caused regression and rejection of tumors. The antitumor effect appeared to involve tumor-specific CTL and protective immunological memory. PT-100 treatment of WEHI 164-inoculated mice increased mRNA expression of cytokines and chemokines known to promote T-cell priming and chemoattraction of T cells and innate effector cells. The role of innate activity was further implicated by observation of significant, although reduced, inhibition of WEHI 164 and A20/2J tumors in immunodeficient mice. PT-100 also demonstrated ability to augment antitumor activity of rituximab and trastuzumab in xenograft models of human CD20(+) B-cell lymphoma and HER-2(+) colon carcinoma where antibody-dependent cytotoxicity can be mediated by innate effector cells responsive to the cytokines and chemokines up-regulated by PT-100. Although CD26/DPP-IV is a potential target for PT-100 in the immune system, it appeared not to be involved because antitumor activity and stimulation of cytokine and chemokine production was undiminished in CD26(-/-) mice.

Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies.

In hematopoiesis, cytokine levels modulate blood cell replacement, self-renewal of stem cells, and responses to disease. Feedback pathways regulating cytokine levels and targets for therapeutic intervention remain to be determined. Amino boronic dipeptides are orally bioavailable inhibitors of dipeptidyl peptidases. Here we show that the high-affinity inhibitor Val-boro-Pro (PT-100) can stimulate the growth of hematopoietic progenitor cells in vivo and can accelerate neutrophil and erythrocyte regeneration in mouse models of neutropenia and acute anemia. Hematopoietic stimulation by PT-100 correlated with increased cytokine levels in vivo. In vitro, PT-100 promoted the growth of primitive hematopoietic progenitor cells by increasing granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-11 production by bone marrow stromal cells. Two molecular targets of PT-100 are expressed by stromal cells- CD26/DPP-IV and the closely related fibroblast activation protein (FAP). Because PT-100 was active in the absence of CD26, FAP appears to be the hematopoietic target for PT-100. Interaction of PT-100 with the catalytic site seems to be required because amino-terminal acetylation of PT-100 abrogated enzyme inhibition and hematopoietic stimulation. PT-100 is a therapeutic candidate for the treatment of neutropenia and anemia. The data support increasing evidence that dipeptidyl peptidases can regulate complex biologic systems by the proteolysis of signaling peptides.