Supplementary Methods, Figures S1-S7 Suppl. Fig. S1, Differentiation of hematopoietic stem cells (CD133+/CD34+) into immature megakarytocytes Suppl. Fig. S2, T-DM1 does not directly induce platelet aggregation in washed platelets Suppl. Fig. S3, At high concentrations, DM1 inhibits agonist-induced platelet aggregation in platelet-rich plasma Suppl. Fig. S4, DM1 conjugates alter the morphology of megakaryocytes Suppl. Fig. S5, Uptake of T-[3H]DM1 varies by differentiation stage Suppl. Fig. S6, HER2 is not expressed on megakaryocytes or platelets Suppl. Fig. S7, Prolonged exposure to T-DM1 results in disruption of cytoskeletal structure in maturing megakaryocytes

Differentially expressed genes in T-DM1-resistant KPL-4 cells with respect to the parental cell line (fold change > 2-fold and P < 0.05 of individual probes for a given gene in the microarray)

Supplemental Figure 1. Upregulated BHLHE41 in KPL-4 TR and BT-474M1 TR cells does not confer resistance to T-DM1. Supplemental Figure 2. Flow cytometry analysis of HER2 and MDR1 expression in KPL-4 P and TR cells, and tumor derived KPL-4 TR cells. Supplemental Figure 3. qRT-PCR analysis of ErbB receptors and ligands in KPL-4 and BT-474M1 P and TR cells. Supplemental Figure 4. Flow cytometry analysis of HER2 expression in BT-474M1 P and TR cells. Supplemental Figure 5. Immunofluorescence detection of cell surface HER2 on BT-474M1 and T-DM1 resistant cells. Supplemental Figure 6. Analysis of T-DM1 uptake in BT-474M1 P and TR, and KPL-4 P and TR cells using radiolabeled T-DM1. Supplemental Figure 7. Treatment of KPL-4 TR cells with anti-IGF-1R antibody 10H5 or the selective c-met inhibitor PHA665725 does not restore sensitivity to T-DM1. Supplemental Figure 8. Upregulated EGFR does not mediate resistance to T-DM1 in KPL-4 TR cells. Supplemental Figure 9. Potential autocrine receptor-ligand interactions for T-DM1 resistance. Supplemental Figure 10. Comparable shRNA knockdown of PTEN in BT-474M1 cells in 2 different shRNA clones, #1 and #3. Supplemental Figure 11. Molecular alterations in BT-474M1 TR cells that did not confer T-DM1 resistance. Supplemental Figure 12. Expression of drug resistance transporters, assessed by qRT-PCR, in KPL-4 and BT-474M1 parental and TR cells. Supplemental Figure 13. The MDR1 inhibitor XR9051 and the BCRP inhibitor Ko143 do not affect cell viability Supplemental Figure 14. MRP4 siRNA in BT-474M1 TR does not reverse T-DM1 resistance. Supplemental Figure 15. MRP4 siRNA in KPL-4 TR does not reverse T-DM1 resistance. Supplemental Figure 16. Expression of SLC46A3 in KPL-4 parental and TR cells assessed by qRT-PCR. Supplemental Figure 17. SLC46A3 siRNA in BT-474M1 TR cells significantly reverses T-DM1 resistance.

BT-474M1 TR vs. Parental cells, gene expression changes >5-fold

KPL-4 TR vs. Parental cells, gene expression changes > 5-fold

Differentially expressed genes in T-DM1-resistant BT-474M1 cells with respect to the parental cell line (fold change > 2 fold and P < 0.05 of individual probes for a given gene in the microarray)

Large molecules therapeutics, also referred to as biologics or biotherapeutics, are proteins designed to modulate their target(s) pharmacology to achieve therapeutic effect. Due to high target specificity, off-target toxicity is rare following the administration of large molecule therapeutics, and (unlike their small molecule counterparts) observed toxic events are more frequently associated with exaggerated pharmacology. As most large molecule therapeutics developed to date are antibodies or antibody-based therapeutics, we review here the salient factors that govern their ADME properties including include charge, hydrophobicity, glycosylation, FcRn affinity, target biology, immunogenicity, and subject physiology as well as well as how the intended pharmacology impacts the design and development of these modalities to maximize therapeutic benefit. In many cases, these principles can also be extended to other biotherapeutic products, including vaccines, gene and cell therapy, tissue, and other proteins that are emerging as therapeutic alternatives in indications of high unmet needs that are briefly described in this chapter.

Disrupted tau proteostasis and transneuronal spread is a pathological hallmark of Alzheimer’s disease. Neurodegenerative diseases remain an unmet medical need and novel disease modifying therapeutics are paramount. Our objective was to develop a mechanistic mathematical model to enhance our understanding of tau antibody pharmacokinetics and pharmacodynamics in animals and humans. A physiologically-based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling approach was employed to support the preclinical development and clinical translation of therapeutic antibodies targeting tau for the treatment of Alzheimer’s disease. The pharmacokinetics of a tau antibody was evaluated in rat and non-human primate microdialysis studies. Model validation for humans was performed using publicly available clinical data for gosuranemab. In-silico analyses were performed to predict tau engagement in human brain for a range of tau antibody affinities and various dosing regimens. PBPK-PD modeling enabled a quantitative understanding for the relationship between dose, affinity, and target engagement, which supported lead candidate optimization and predictions of clinically efficacious dosing regimens.

There are several antibody therapeutics in preclinical and clinical development, industry-wide, for the treatment of central nervous system (CNS) disorders. Due to the limited permeability of antibodies across brain barriers, the quantitative understanding of antibody exposure in the CNS is important for the design of antibody drug characteristics and determining appropriate dosing regimens. We have developed a minimal physiologically-based pharmacokinetic (mPBPK) model of the brain for antibody therapeutics, which was reduced from an existing multi-species platform brain PBPK model. All non-brain compartments were combined into a single tissue compartment and cerebral spinal fluid (CSF) compartments were combined into a single CSF compartment. The mPBPK model contains 16 differential equations, compared to 100 in the original PBPK model, and improved simulation speed approximately 11-fold. Area under the curve ratios for minimal versus full PBPK models were close to 1 across species for both brain and plasma compartments, which indicates the reduced model simulations are similar to those of the original model. The minimal model retained detailed physiological processes of the brain while not significantly affecting model predictability, which supports the law of parsimony in the context of balancing model complexity with added predictive power. The minimal model has a variety of applications for supporting the preclinical development of antibody therapeutics and can be expanded to include target information for evaluating target engagement to inform clinical dose selection.

The Port Delivery System with ranibizumab (PDS) is an investigational drug delivery system designed to provide continuous intravitreal release of ranibizumab for extended durations. The PDS consists of a permanent, surgically placed, refillable intraocular implant; a customized formulation of ranibizumab; and ancillary devices to support surgery and refill procedures. A toxicology program was conducted to evaluate the ocular toxicology and biocompatibility of the PDS to support its clinical development program and product registrational activities. PDS safety studies included a 6-month chronic toxicology evaluation in minipigs as well as evaluation of nonfunctional surrogate implants (comprised of the same implant materials but without ranibizumab) in rabbits. Biocompatibility of the implant and ancillary devices was evaluated in both in vitro and in vivo studies. Implants and extracts from implants and ancillary devices were nongenotoxic, noncytotoxic, nonsensitizing, and nonirritating. Ocular findings were comparable between implanted and sham-operated eyes, and no systemic toxicity was observed. The results of this nonclinical toxicology program demonstrated that the PDS was biocompatible and that intravitreal delivery of ranibizumab via the PDS did not introduce any new toxicology-related safety concerns relative to intravitreal injections, supporting ongoing PDS clinical development and product registrational evaluation.

PURPOSE:Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody-drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu.METHODS:Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo.RESULTS:Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected.CONCLUSIONS:These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

A phage-derived human monoclonal antibody against VEGF-C was developed as a potential anti-tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti-VEGF-C to determine potential protein and cell-based interactions with the antibody as well as any matrix-dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti-VEGF-C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF-C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.

Background and PurposePolatuzumab vedotin is an antibody–drug conjugate (ADC) being developed for non‐Hodgkin's lymphoma. It contains a humanized anti‐CD79b IgG1 monoclonal antibody linked to monomethyl auristatin E (MMAE), an anti‐mitotic agent. Polatuzumab vedotin binds to human CD79b only. Therefore, a surrogate ADC that binds to cynomolgus monkey CD79b was used to determine CD79b‐mediated pharmacological effects in the monkey and to enable first‐in‐human clinical trials.Experimental ApproachPolatuzumab vedotin, the surrogate ADC, and the corresponding antibodies were evaluated in different assays in vitro and in animals. In vitro assessments included binding to peripheral blood mononuclear cells from different species, binding to a human and monkey CD79b‐expressing cell line, binding to human Fcγ receptors, and stability in plasma across species. In vivo, ADCs were assessed for anti‐tumour activity in mice, pharmacokinetics/pharmacodynamics in monkeys, and toxicity in rats and monkeys.Key ResultsPolatuzumab vedotin and surrogate ADC bind with similar affinity to human and cynomolgus monkey B cells, respectively. Comparable in vitro plasma stability, in vivo anti‐tumour activity, and mouse pharmacokinetics were also observed between the surrogate ADC and polatuzumab vedotin. In monkeys, only the surrogate ADC showed B‐cell depletion and B‐cell‐mediated drug disposition, but both ADCs showed similar MMAE‐driven myelotoxicity, as expected.Conclusions and ImplicationsThe suitability of the surrogate ADC for evaluation of CD79b‐dependent pharmacology was demonstrated, and anti‐tumour activity, pharmacokinetics/pharmacodynamics, and toxicity data with both ADCs supported the entry of polatuzumab vedotin into clinical trials.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

Monoclonal antibodies (mAbs), which are now more frequently administered by subcutaneous (SC) injection rather than intravenously, have become a tremendously successful drug format across a wide range of therapeutic areas. Preclinical evaluations of mAbs to be administered by SC injection are typically performed in species such as mice, rats, minipigs, and cynomolgus monkeys to obtain critical information regarding formulation performance and prediction of PK/PD outcomes needed to select clinical doses for first-in-human studies. Despite extensive efforts, no preclinical model has been identified to date that accurately predicts clinical outcomes for these SC injections. We have addressed this deficiency with a novel in vitro instrument, termed Scissor, to model events occurring at the SC injection site and now further validated this approach using a set of eight mAbs for which clinical PK/PD outcomes have been obtained. Diffusion of these mAbs from the Scissor system injection cartridge into a large volume physiological buffer, used to emulate mAb movement from the SC injection site into the systemic circulation, provided distinct profiles when monitored over a 6 h period. Curve-fitting analysis of these profiles using the Hill equation identified parameters that were used, along with physicochemical properties for each mAb, in a partial least squares analysis to define a relationship between molecule and formulation properties with clinical PK outcomes. The results demonstrate that parameters of protein charge at neutral pH and isoelectric point (pI) along with combined formulation properties such as viscosity and mAb concentration can dictate the movement of the mAb from the injection cartridge to infinite sink compartment. Examination of profile characteristics of this movement provided a strong predictive correlation for these eight mAbs. Together, this approach demonstrates the feasibility of this in vitro modelling strategy as a tool to identify drug and formulation properties that can define the performance of SC injected medicines and provide the potential for predicting clinical outcomes that could be useful for formulation selection and a first-in-human clinical dosing strategy.