Targeting the 5T4 oncofetal glycoprotein with an antibody drug conjugate (A1mcMMAF) improves survival in patient-derived xenograft models of acute lymphoblastic leukemia.

Outcome in childhood acute lymphoblastic leukemia is prognosticated from levels of minimal residual disease after remission induction therapy. Higher levels of minimal residual disease are associated with inferior results even with intensification of therapy, thus suggesting that identification and targeting of minimal residual disease cells could be a therapeutic strategy. Here we identify high expression of 5T4 in subclonal populations of patient-derived xenografts from patients with high, post-induction levels of minimal residual disease. 5T4-positive cells showed preferential ability to overcome the NOD-scidIL2Rγnull mouse xenograft barrier, migrated in vitro on a CXCL12 gradient, preferentially localized to bone marrow in vivo and displayed the ability to reconstitute the original clonal composition on limited dilution engraftment. Treatment with A1mcMMAF (a 5T4-antibody drug conjugate) significantly improved survival without overt toxicity in mice engrafted with a 5T4-positive acute lymphoblastic leukemia cell line. Mice engrafted with 5T4-positive patient-derived xenograft cells were treated with combination chemotherapy or dexamethasone alone and then given A1mcMMAF in the minimal residual disease setting. Combination chemotherapy was toxic to NOD-scidIL2Rγnull mice. While dexamethasone or A1mcMMAF alone improved outcomes, the sequential administration of dexamethasone and A1mcMMAF significantly improved survival (P=0.0006) over either monotherapy. These data show that specifically targeting minimal residual disease cells improved outcomes and support further investigation of A1mcMMAF in patients with high-risk B-cell precursor acute lymphoblastic leukemia identified by 5T4 expression at diagnosis.

A general approach to site-specific antibody drug conjugates.

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads.

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Preclinical Development of an anti-5T4 Antibody-Drug Conjugate: Pharmacokinetics in Mice, Rats, and NHP and Tumor/Tissue Distribution in Mice.

The pharmacokinetics of an antibody (huA1)-drug (auristatin microtubule disrupting MMAF) conjugate, targeting 5T4-expressing cells, were characterized during the discovery and development phases in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multidose toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the released payload using an LC-MS/MS method. In addition, the distribution of the Ab, ADC, and released payload (cys-mcMMAF) was determined in a number of tissues (tumor, lung, liver, kidney, and heart) in two tumor mouse models (H1975 and MDA-MB-361-DYT2 models) using similar LBA and LC-MS/MS methods. Tissue distribution studies revealed preferential tumor distribution of cys-mcMMAF and its relative specificity to the 5T4 target containing tissue (tumor). Single dose studies suggests lower CL values at the higher doses in mice, although a linear relationship was seen in cynomolgus monkeys at doses from 0.3 to 10 mg/kg with no evidence of TMDD. Evaluation of DAR (drug-antibody ratio) in cynomolgus monkeys (at 3 mg/kg) indicated that at least half of the payload was still on the ADC 1 to 2 weeks after IV dosing. After multiple doses, the huA1 and conjugate data in rats and monkeys indicate that exposure (AUC) increases with increasing dose in a linear fashion. Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose, although exposure was very low and its pharmacokinetics appeared to be formation rate limited. The incidence of ADA was generally low in rats and monkeys. We will discuss cross species comparison, relationships between the Ab, ADC, and released payload exposure after multiple dosing, and insights into the distribution of this ADC with a focus on experimental design as a way to address or bypass apparent obstacles and its integration into predictive models.

Phenotype of TPBG Gene Replacement in the Mouse and Impact on the Pharmacokinetics of an Antibody-Drug Conjugate.

The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody-drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics.

Evolving Strategies for Target Selection for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) represent a promising modality for the treatment of cancer. The therapeutic strategy is to deliver a potent drug preferentially to the tumor and not normal tissues by attaching the drug to an antibody that recognizes a tumor antigen. The selection of antigen targets is critical to enabling a therapeutic window for the ADC and has proven to be surprisingly complex. We surveyed the tumor and normal tissue expression profiles of the targets of ADCs currently in clinical development. Our analysis demonstrates a surprisingly broad range of expression profiles and the inability to formalize any optimal parameters for an ADC target. In this context, we discuss additional considerations for ADC target selection, including interdependencies among biophysical properties of the drug, biological functions of the target and strategies for clinical development. The TPBG (5T4) oncofetal antigen and the anti-TPBG ADC A1-mcMMAF are highlighted to demonstrate the relevance of the target's biological function. Emerging platform technologies and novel biological insights are expanding ADC target space and transforming strategies for target selection.

Mediation of antitumor activity by AZD4820 oncolytic vaccinia virus encoding IL-12.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

On translation of antibody drug conjugates efficacy from mouse experimental tumors to the clinic: a PK/PD approach.

Objectives of the present investigation were: (1) to compare three literature reported tumor growth inhibition (TGI) pharmacodynamic (PD) models and propose an optimal new model that best describes the xenograft TGI data for antibody drug conjugates (ADC), (2) to translate efficacy of the ADC Trastuzumab-emtansine (T-DM1) from mice to patients using the optimized PD model, and (3) to apply the translational strategy to predict clinically efficacious concentrations of a novel in-house anti-5T4 ADC, A1mcMMAF. First, the performance of all four of the PD models (i.e. 3 literature reported + 1 proposed) was evaluated using TGI data of T-DM1 obtained from four different xenografts. Based on the estimates of the pharmacodynamic/pharmacokinetic (PK/PD) modeling, a secondary parameter representing the efficacy index of the drug was calculated, which is termed as the tumor static concentration (TSC). TSC values derived from all four of the models were compared with each other, and with literature reported values, to assess the performance of these models. Subsequently, using the optimized PK/PD model, PD parameters obtained from different cell lines, human PK, and the proposed translational strategy, clinically efficacious doses of T-DM1 were projected. The accuracy of projected efficacious dose range for T-DM1 was verified by comparison with the clinical doses. Aforementioned strategy was then applied to A1mcMMAF for projecting its efficacious concentrations in clinic. TSC values for A1mcMMAF, obtained by fitting TGI data from 4 different xenografts with the proposed PK/PD model, were estimated to range from 0.6 to 11.5 µg mL⁻¹. Accordingly, the clinically efficacious doses for A1mcMMAF were projected retrospectively. All in all, the improved PD model and proposed translational strategy presented here suggest that appropriate correction for the clinical exposure and employing the TSC criterion can help translate mouse TGI data to predict first in human doses of ADCs.

Advances in antibody-based therapy in oncology.

Monoclonal antibodies are a growing class of targeted cancer therapeutics, characterized by exquisite specificity, long serum half-life, high affinity and immune effector functions. In this review, we outline key advances in the field with a particular focus on recent and emerging classes of engineered antibody therapeutic candidates, discuss molecular structure and mechanisms of action and provide updates on clinical development and practice.

Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.

Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade.

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

Potent and sustained inhibition of HIF-1α and downstream genes by a polyethyleneglycol-SN38 conjugate, EZN-2208, results in anti-angiogenic effects.

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFß1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208's superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.

Targeting and pharmacology of an anti-IL13Rα2 antibody and antibody-drug conjugate in a melanoma xenograft model.

IL13Rα2 is a cell surface tumor antigen that is overexpressed in multiple tumor types. Here, we studied biodistribution and targeting potential of an anti-IL13Rα2 antibody (Ab) and anti-tumor activity of anti-IL13Rα2-antibody-drug conjugate (ADC). The anti-IL13Rα2 Ab was labeled with fluorophore AF680 or radioisotope 89Zr for in vivo tracking using fluorescence molecular tomography (FMT) or positron emission tomography (PET) imaging, respectively. Both imaging modalities showed that the tumor was the major uptake site for anti-IL13Rα2-Ab, with peak uptake of 5-8% ID and 10% ID/g as quantified from FMT and PET, respectively. Pharmacological in vivo competition with excess of unlabeled anti-IL13Rα2-Ab significantly reduced the tumor uptake, indicative of antigen-specific tumor accumulation. Further, FMT imaging demonstrated similar biodistribution and pharmacokinetic profiles of an auristatin-conjugated anti-IL13Rα2-ADC as compared to the parental Ab. Finally, the anti-IL13Rα2-ADC exhibited a dose-dependent anti-tumor effect on A375 xenografts, with 90% complete responders at a dose of 3 mg/kg. Taken together, both FMT and PET showed a favorable biodistribution profile for anti-IL13Rα2-Ab/ADC, along with antigen-specific tumor targeting and excellent therapeutic efficacy in the A375 xenograft model. This work shows the great potential of this anti-IL13Rα2-ADC as a targeted anti-cancer agent.

NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells.

Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

PF-06804103, A Site-specific Anti-HER2 Antibody-Drug Conjugate for the Treatment of HER2-expressing Breast, Gastric, and Lung Cancers.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers.

PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Marked therapeutic efficacy of a novel polyethylene glycol-SN38 conjugate, EZN-2208, in xenograft models of B-cell non-Hodgkin's lymphoma.

Examination of the clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active metabolite of CPT-11, has not been possible to date due to poor solubility of SN38. Here we evaluated the activity of EZN-2208, a water-soluble polyethyleneglycol-SN38 conjugate, in pre-clinical models of Burkitt's non-Hodgkin's lymphoma (NHL) (Raji and Daudi), and follicular NHL (DoHH2). In vitro, the IC50 of EZN-2208 ranged from 3-24 nM, which was 30- to 45-fold lower than CPT-11 or 2.5- to 3.5-fold higher than SN38. In both an early-disease Raji model and an advanced-disease Daudi model, treatment with multiple doses of EZN-2208 resulted in 90% and 100% cures of animals, respectively (cure defined as no sign of tumors by gross observations at the termination of study). The activity of EZN-2208 was dramatically superior to that of CPT-11 in all three models. The excellent therapeutic efficacy of EZN-2208 in several B-cell NHL xenograft models merits its evaluation in the clinic for lymphoid malignancies.

Novel delivery of SN38 markedly inhibits tumor growth in xenografts, including a camptothecin-11-refractory model.

PURPOSE: Clinical development of SN38, the active metabolite of camptothecin-11 (CPT-11), has been hampered due to its poor solubility. We have developed a novel polymer-drug conjugate, EZN-2208, made by linking SN38 with a multiarm polyethylene glycol via a glycine linker. EXPERIMENTAL DESIGN: The in vitro cytotoxicity of EZN-2208 was tested using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. The therapeutic efficacy of EZN-2208 was evaluated in various xenografts, including an in vivo-selected CPT-11-refractory model. Tumor and blood concentration of EZN-2208, CPT-11, and SN38 was determined by high-performance liquid chromatography. RESULTS: In vitro, EZN-2208 was 10- to 245-fold more potent than CPT-11 in a panel of human tumor cell lines. In xenograft models of MX-1 breast, MiaPaCa-2 pancreatic, or HT-29 colon carcinoma, treatment with either a single dose or multiple injections of EZN-2208 was more efficacious (and in some cases produced tumor eradication for >16 weeks) compared with CPT-11 at their respective maximum tolerated doses or corresponding dose levels (P < 0.01). Most interestingly, EZN-2208 showed marked antitumor activity in animals that developed resistance to an 8-day course of CPT-11 treatment, as well as outperformed CPT-11 as second-round therapy in mice initially sensitive to CPT-11. EZN-2208 had prolonged circulation in the blood compared with CPT-11, resulting in high tumor exposure. This resulted in higher and longer-lasting tumor exposure of free SN38 in mice given EZN-2208 compared with those given CPT-11. CONCLUSIONS: Preclinical data suggest that EZN-2208 may be a promising anticancer agent in a wide variety of clinical settings, including tumors refractory to CPT-11 treatment.