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Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg483↓Val484) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg483-Val484 with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON–Fc fusion protein, whereas hepsin-cleaved MSP bound with a KD of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders. Mol Cancer Res; 9(9); 1175–86. ©2011 AACR.

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Integrin-β6 (ITGB6) is a transmembrane protein that partners exclusively with the integrin-αV subunit to form the integrin-αVβ6 heterodimer. Due to its roles in tissue remodeling and cancer pathogenesis, therapeutic targeting of integrin-αVβ6 has been investigated for both fibrotic diseases and cancers. ITGB6 is homogenously overexpressed in numerous solid tumor types, including breast, head and neck, pancreatic, and non-small cell lung cancers. Additionally, its expression is an unfavorable prognostic marker in multiple cancers. However, past attempts to therapeutically target ITGB6 have been hindered by its expression in several normal tissues, including bladder, skin, lung, kidney, and muscle. For example, clinical testing of an integrin αVβ6-targeting monoclonal antibody was discontinued reportedly due to toxicity concerns. To reduce on-target/off-tumor toxicities from ITGB6 expression in normal tissues and to enable safe targeting of ITGB6 in solid tumors, we developed an ITGB6-targeting ProTriTAC™ (Protease-activated Tri-specific T cell Activating Construct), a T cell engager prodrug which is engineered to be preferentially activated in tumor tissue. ProTriTACs consists of three humanized antibody-derived binding domains on a single polypeptide chain: anti-albumin for half-life extension, anti-CD3 for T cell engagement, and anti-target antigen for tumor cell engagement. The anti-albumin domain, bearing a masking moiety and a protease-cleavable linker, keeps the prodrug inert by inhibiting binding of the adjacent anti-CD3 domain to T cells. Cleavage of the linker by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potent active drug that directs T cell killing within the tumor. As designed, in vitro binding and functional assays show that the protease-activated T cell engager has >1000-fold improved binding to human T cells and ~100-fold improved T cell killing compared to the intact masked prodrug. In immunodeficient mice reconstituted with human T cells, anti-ITGB6 ProTriTAC demonstrated robust anti-tumor activity in multiple ITGB6-expressing xenograft tumor models, including HCC70 (breast), CAL27 (head and neck), and HPAFII (pancreatic), with complete tumor regression as low as 30 μg/kg. To determine the potential toxicity and toxicokinetic characteristics of anti-ITGB6 ProTriTAC, an intra-animal dose escalation was conducted in cynomolgus monkeys. Starting at 20 μg/kg with weekly 3-fold dose escalations, anti-ITGB6 ProTriTAC was well tolerated up to 540 μg/kg, which was the highest dose tested. The preclinical activity in rodent tumor models, coupled to its tolerability in cynomolgus monkeys, support the development of anti-ITGB6 ProTriTAC as a therapeutic in a broad range of ITGB6-expressing solid tumors. Citation Format: Regina J. Lin, Sony S. Rocha, Maria R. Dayao, Subramanian Thothathri, Raphaela Rose Banzon, Kevin J. Wright, Wade Aaron, Yinghua Xiao, Nick Bergo, Linh To, Mabel Bush, Manasi Barath, Yi Yang, Timothy Yu, Willis Kwong, Hubert Situ, Eric Bragg, Jessica O'Rear, Kevin Carlin, Stephen Yu, Maritza Solorio, Bryan Lemon, Richard Austin, Holger Wesche, S. Jack Lin. ITGB6 ProTriTAC™, a protease-activated T cell engager prodrug targeting Integrin-β6 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2927.

TriTAC® is a T cell engager platform comprised of three binding domains: a target binder for tumor cell engagement, an albumin binder for half-life extension, and a CD3 binder for T cell engagement. HPN328 is a Delta-like ligand 3 (DLL3)-targeting TriTAC currently being evaluated in a Phase 1/2 clinical trial enrolling patients with advanced cancers associated with DLL3 expression, including small cell lung cancer (SCLC) and other neuroendocrine malignancies (NCT04471727). While HPN328 has demonstrated single agent clinical activity in relapsed/refractory SCLC patients, combinations with other immunotherapeutic agents could further potentiate HPN328 anti-tumor activity and enable the testing of HPN328 in earlier lines of therapy. To test this hypothesis, we developed a human CD3ε (hCD3ε) immunocompetent mouse model that has the epitope of human CD3ε, as recognized by TriTAC molecules, specifically knocked-in (KI) to the mouse CD3ε gene. hCD3ε KI mice have comparable immune cell repertoire as wild-type mice. However, unlike wild-type mice, TriTAC molecules bind to the T cells in hCD3ε KI mice. In a MC38 tumor model, an EGFR-targeting TriTAC was able to exert the expected anti-tumor activity and EGFR-dependent toxicity, thereby confirming TriTACs can engage and activate endogenous T cells in this immunocompetent hCD3ε KI mouse model. Next, we tested HPN328 in hCD3ε KI mice with MC38 tumors that have been engineered to express human DLL3. As expected, HPN328 inhibited tumor growth in a dose-dependent manner. HPN328 also resulted in a notable upregulation of PD-L1 in these tumors, suggesting that HPN328 in combination with an anti-PD-L1 antibody could potentially drive deeper anti-tumor responses. Indeed, sub-therapeutic doses of HPN328 in combination with an anti-PD-L1 antibody enhanced the anti-tumor activity of HPN328 when compared to either treatment alone. Atezolizumab and durvalumab, anti-PD-L1 antibodies, are approved in combination with platinum-doublet chemotherapy as part of front-line therapy for extensive-stage SCLC. These results demonstrate the utility of combining anti-PD-L1 antibodies to enhance the anti-tumor activity of HPN328 and further supports investigation of this combinatorial approach in patients. Clinical studies of HPN328 in combination with atezolizumab are planned. Citation Format: Laura B. Valenzuela, Chi-Heng Wu, Wade Aaron, Raphalea R. Banzon, Mabel Bush, S. Jack Lin, Sony S. Rocha, Subramanian Thothathri, Holger Wesche, Mary Ellen Molloy, Banmeet S. Anand. Anti-tumor activity of HPN328, a DLL3-targeting tri-specific, half-life extended T cell engager, is enhanced by combining with an anti-PD-L1 antibody in an immunocompetent mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5070.

Tumor-associated calcium signal transducer 2 (Trop2) is a cell surface glycoprotein that promotes cell renewal, proliferation and tumorigenesis. Trop2 is homogenously overexpressed in numerous solid tumor types, including breast, head and neck, prostate, bladder, and non-small cell carcinoma; however, Trop2 is also expressed in several normal tissues, including skin, respiratory tract, and female reproductive organs. Although Trop2-targeted antibody drug conjugates (ADCs) have demonstrated clinical activity, with regulatory approvals in subsets of breast and bladder cancers, their broader utility is still hampered by off-target payload-mediated toxicities. To address this issue, and to avoid on-target off-tumor toxicities, we developed a Trop2-targeting ProTriTAC™ (Protease-activated Tri-specific T cell Activating Construct), a T cell engager prodrug which is engineered to be preferentially activated in tumor tissue. ProTriTACs consists of three humanized antibody-derived binding domains on a single polypeptide chain: anti-albumin for half-life extension, anti-CD3 for T cell engagement, and anti-target antigen for tumor cell engagement. The anti-albumin domain, bearing a masking moiety and a protease-cleavable linker, keeps the prodrug inert by inhibiting binding of the adjacent anti-CD3 domain to T cells. Cleavage of the linker by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potent active drug that directs T cell killing within the tumor. As designed, in vitro binding and functional assays show that the protease-activated T cell engager has >1000-fold improved binding to primary human T cells and ~100-fold improved T cell killing compared to its intact masked prodrug. In immunodeficient mice reconstituted with human T cells, anti-Trop2 ProTriTAC demonstrated robust anti-tumor activity in multiple xenograft tumor models, including HCC70 (breast), CAL27 (head and neck), and HPAFII (pancreatic), with complete tumor regression as low as 30 μg/kg. To determine the potential toxicity and toxicokinetic characteristics of anti-Trop2 ProTriTAC, an intra-animal dose escalation was conducted in cynomolgus monkeys until individual maximum tolerated dose (MTD). The starting dose was 20 μg/kg with weekly 3-fold dose escalations until MTD. The one-month study revealed that anti-Trop2 ProTriTAC was tolerated at the 180 μg/kg dose level, but not at the next higher dose level of 540 μg/kg. Toxicokinetics, gross pathology, histopathology, and cytokine data will be presented. The preclinical activity in rodent tumor models, coupled to its tolerability in cynomolgus monkeys, support the development of anti-Trop2 ProTriTAC as a therapeutic in a broad range of Trop2-expressing solid tumors. Citation Format: Sony S. Rocha, Regina Lin, Maria R. Dayao, Raphaela Rose Banzon, Subramanian Thothathri, Kevin J. Wright, Wade Aaron, Yinghua Xiao, Nick Bergo, Linh To, Mabel Bush, Manasi Barath, Timothy Yu, Willis Kwong, Hubert Situ, Eric Bragg, Jessica O'Rear, Kevin Carlin, Stephen Yu, Maritza Solorio, Bryan Lemon, Richard Austin, Holger Wesche, S. Jack Lin. TROP2 ProTriTAC™, a protease-activated T cell engager prodrug targeting TROP2 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2928.

BackgroundMasking serves an important role in reducing the transmission of respiratory viruses, including SARS-CoV-2. During the COVID-19 pandemic, several perspective and review articles have also argued that masking reduces the risk of developing severe disease by reducing the inoculum dose received by the contact. This hypothesis – known as the ‘variolation hypothesis’ – has gained considerable traction since its development.MethodsTo assess the plausibility of this hypothesis, we develop a quantitative framework for understanding the relationship between (i) inoculum dose and the risk of infection and (ii) inoculum dose and the risk of developing severe disease. We parameterize the mathematical models underlying this framework with parameters relevant for SARS-CoV-2 to quantify these relationships empirically and to gauge the range of inoculum doses in natural infections. We then identify and analyze relevant experimental studies of SARS-CoV-2 to ascertain the extent of empirical support for the proposed framework.ResultsMathematical models, when simulated under parameter values appropriate for SARS-CoV-2, indicate that the risk of infection and the risk of developing severe disease both increase with an increase in inoculum dose. However, the risk of infection increases from low to almost certain infection at low inoculum doses (with 6 initially infected cells. By drawing on studies that have estimated transmission bottleneck sizes of SARS-CoV-2, we find that inoculum doses are low in natural SARS-CoV-2 infections. As such, reductions in inoculum dose through masking or greater social distancing are expected to reduce the risk of infection but not the risk of developing severe disease conditional on infection. Our review of existing experimental studies support this finding.ConclusionsWe find that masking and other measures such as distancing that act to reduce inoculum doses in natural infections are highly unlikely to impact the contact’s risk of developing severe disease conditional on infection. However, in support of existing empirical studies, we find that masking and other mitigation measures that reduce inoculum dose are expected to reduce the risk of infection with SARS-CoV-2. Our findings therefore undermine the plausibility of the variolation hypothesis, underscoring the need to focus on other factors such as comorbidities and host age for understanding the heterogeneity in disease outcomes for SARS-CoV-2.

Social decision-making requires the integration of reward valuation and social cognition systems, both dependent on the orbitofrontal cortex (OFC). How these two OFC functions interact is largely unknown. We recorded intracranial activity from the OFC of ten patients making choices in the context of different types of inequity (disadvantageous vs. advantageous). We found that high-frequency activity (70-150 HZ) encoded the amount of self-reward, consistent with previous reports. We also observed novel evidence for encoding in human OFC of the social counterpart’s reward as well as the type of inequity being experienced. Additionally, we find social context modulates reward encoding: depending on inequity type, reward encoding was switched on and off rapidly within electrodes, across trials. These results provide direct evidence for explicit encoding of self- and other- rewards in the human OFC, and for rapid and reversible changes in encoding schemes driven by socially relevant contexts.

Sleep oscillations provide a key substrate to facilitate memory processing, the underlying mechanism of which may involve the overnight homeostatic regulation of plasticity at a synaptic and whole-network level. However, there remains a lack of human data demonstrating if and how sleep enhances memory consolidation and associated neural homeostasis. We combined intracranial recordings and scalp electroencephalography (EEG) in humans to reveal a new role for rapid eye movement (REM) sleep in promoting the homeostatic recalibration of optimal excitation/inhibition-balance. Moreover, the extent of this REM-sleep homeostatic recalibration predicted the success of overnight memory consolidation, expressly the modulation of hippocampal— neocortical excitability favoring remembering rather than forgetting. The findings describe a novel, fundamental role of human REM sleep in maintaining neural homeostasis, thereby enhancing long-term memory.

Objective To report a case of untreated classic 21 hydroxylase (OH) deficiency congenital adrenal hyperplasia (CAH) in a transgender patient resulting in pulmonary embolisms (PEs) and bilateral adrenal masses. Methods A 36-year-old male (birth sex: female) presenting with bilateral PEs in the setting of long-standing, untreated classic 21OH CAH was also found to have bilateral adrenal masses (unconfirmed myelolipomas). Results Further history revealed a known diagnosis of CAH. The patient had been treated with glucocorticoid and mineralocorticoid replacement in childhood but stopped taking these medications against medical advice. During his hospital admission, he was noted to have elevated 17-hydroxyprogesterone, low cortisol with elevated ACTH levels, and male-level testosterone measurements. CT of the abdomen/pelvis revealed a 23 cm mass in the left renal fossa and a 2.5 cm mass in the right renal fossa consistent with bilateral adrenal myelolipomas. The patient attended follow-up in clinic, but declined any further hormonal treatment as he identified as male and felt further treatment was unnecessary. Conclusion This case demonstrated the unique long-term effects of untreated classic CAH due to 21OH deficiency, including bilateral adrenal myelolipoma, adrenal compensation to the point of producing male-level androgens, and possibly PEs. Treatment with hydrocortisone was recommended to suppress ACTH and it was planned that the patient would eventually start on testosterone (although this would have been complicated by his bilateral PEs). Potential aetiologies for the PEs included vascular compression of the renal artery (which could explain the elevated EPO/erythrocytosis contributing to hypercoagulability) or the renal vein by the adrenal mass. LEARNING POINTS Gender dysphoria in patients with congenital adrenal hyperplasia (CAH) is not uncommon. Adrenal enlargement can allow untreated CAH patients to compensate. Pulmonary embolisms can be a consequence of treating as well as untreated CAH.

Sleep is indispensable for most animals’ cognitive functions, and is hypothesized to be a major factor in memory consolidation. Although we do not fully understand the mechanisms of network reorganisation driving memory consolidation, available data suggests that sleep-associated neurochemical changes may be important for such processes. In particular, global acetylcholine levels change across the sleep/wake cycle, with high cholinergic tone during wake and REM sleep and low cholinergic tone during slow wave sleep. Furthermore, experimental perturbation of cholinergic tone has been shown to impact memory storage. Through in silico modeling of neuronal networks, we show how spiking dynamics change in highly heterogenous networks under varying levels of cholinergic tone, with neuronal networks under high cholinergic modulation firing asynchronously and at high frequencies, while those under low cholinergic modulation exhibit synchronous patterns of activity. We further examined the network’s dynamics and its reorganization mediated via changing levels of acetylcholine within the context of different scale-free topologies, comparing network activity within the hub cells, a small group of neurons having high degree connectivity, and with the rest of the network. We show a dramatic, state-dependent change in information flow throughout the network, with highly active hub cells integrating information in a high-acetylcholine state, and transferring it to rest of the network in a low-acetylcholine state. This result is experimentally corroborated by frequency-dependent frequency changes observed in vivo experiments. Together, these findings provide insight into how new neurons are recruited into memory traces during sleep, a mechanism which may underlie system memory consolidation.

Premature Ovarian Insufficiency (POI) is a loss of ovarian function in women less than 40 years old. We present a case of a female with primary amenorrhea due to autoimmune primary ovarian insufficiency (APOI), in which diagnosis was delayed as she had normal breast and pubic/axillary hair development. A 18-year-old female presented to endocrine clinic for evaluation of primary amenorrhea and hypoplastic uterus and ovaries. During her regular follow ups with her pediatrician it was noted that she had not undergone menarche but since her breast development was normal, further evaluation was not pursued. She had a complicated social history and missed several of her follow up appointments. However, further evaluation was done after she presented with severe pain during intercourse. During her physical exam, it was found she had a vaginal opening of 4 cm with an unpalpable cervix. She did however have pubic/axillary hair and breast development consistent with Tanner stage V. Pelvic MRI showed hypoplastic uterus/ovaries. Her lab work revealed she had a total testosterone of 28ng/dL, prolactin 11.1ng/mL, TSH 1.68iIU/mL, LH 42.2mIU/mL, FSH99.77 mIU/mL, estradiol29.36 pg/ML, AMH 3.1ng/mL. Her karyotype was 46XX. Testing for Fragile×Syndrome and 21 hydroxylase antibodies were negative. Anti-ovarian antibody (AOA) was obtained and found to be positive (1: 160). She was diagnosed with APOI and started on estrogen with a goal to increase to adult doses over 1.5-2 years. Progestins are planned to be introduced once she reaches adult doses of estrogen or develops breakthrough bleeding. Typical presentation of patients with POI is primary amenorrhea and lack of breast development during puberty. However, it is important to remember that there can be a broad spectrum of presenting symptoms with variable breast, uterine, and ovarian development as not all patients have the same degree of estrogen deficiency. This is a case of a patient with POI with hypoplastic uterus and ovaries who had Tanner V breast development. Given this patients ovarian atrophy it is likely that the majority of estrogen production originated from adrenal glands and fat cells, which contributed to the normal development of secondary sexual characteristics delaying her diagnosis. Therefore, identification of abnormal menstrual patterns in adolescence should always raise concern for POI even in the setting of normal breast and pubic/axillary hair development. POI is due to premature deprivation of estrogen with subsequent amenorrhea and compensatory elevations in FSH/LH. Early diagnosis of this condition can prevent potential health complications in such as decreased bone mineral density, psychological complications, and extremely painful intercourse as our patient had. It is important to note that fertility in patients with POI is also unpredictable, however earlier diagnosis/treatment can improve fertility preservation, especially in women with normal AMH levels such as this patient. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The recently discovered neutrophil serine protease 4 (NSP4) is the fourth member of the NSP family, which includes the well-studied neutrophil elastase, proteinase 3 and cathepsin G. Like the other three NSP members, NSP4 is synthesized by myeloid precursors in the bone marrow and, after cleavage of the two-amino acid activation peptide, is stored as an active protease in azurophil granules of neutrophils. Based on its primary amino acid sequence, NSP4 is predicted to have a shallow S1 specificity pocket with elastase-like substrate specificity. However, NSP4 was found to preferentially cleave after an arginine residue. Structural studies resolved this paradox by revealing an unprecedented mechanism of P1-arginine recognition. In contrast to the canonical mechanism in which the P1-arginine residue points down into a deep S1 pocket, the arginine side chain adopts a surface-exposed 'up' conformation in the NSP4 active site. This conformation is stabilized by the Phe190 residue, which serves as a hydrophobic platform for the aliphatic portion of the arginine side chain, and a network of hydrogen bonds between the arginine guanidium group and the NSP4 residues Ser192 and Ser216. This unique configuration allows NSP4 to cleave even after naturally modified arginine residues, such as citrulline and methylarginine. This non-canonical mechanism, characterized by the hallmark 'triad' Phe190-Ser192-Ser216, is largely preserved throughout evolution starting with bony fish, which appeared about 400 million years ago. Although the substrates and physiological role of NSP4 remain to be determined, its remarkable evolutionary conservation, restricted tissue expression and homology to other neutrophil serine proteases anticipate a function in immune-related processes.

CD3-targeted T cell engagers are potent anti-tumor therapies, but their development often requires management of cytokine release syndrome (CRS). One strategy to reduce CRS is subcutaneous dosing, which is hypothesized to mitigate CRS by reducing the maximum drug concentration (Cmax) and preserve efficacy by maintaining the same minimum drug concentration (Cmin) as intravenous dosing. Although promising for mitigating CRS, this approach is limited by its increased immunogenicity risks. A T cell engager designed to be dosed intravenously but engineered to mimic the PK properties of subcutaneous dosing could alleviate CRS without increasing immunogenicity. Here we describe TriTAC-XR, a platform of T cell engager prodrugs designed to become slowly activated in systemic circulation. This extended-release mechanism results in a slow build-up of circulating active drug and minimizes the Cmax/Cmin ratio, similar to subcutaneous dosing. TriTAC-XR prodrugs were engineered by adding a peptide mask and protease-cleavable linker to the N-terminus of a TriTAC, a constitutively active and half-life extended T cell engager. The mask binds to the anti-CD3ε domain and inhibits T cell binding. Upon cleavage by systemic proteases, active T cell engager is released. In vitro, TriTAC-XR has markedly reduced binding to recombinant CD3ε protein and to primary T cells as well as reduced potency in functional T cell-dependent cellular cytotoxicity (TDCC) assays compared to its unmasked active drug. In cynomolgus monkeys, TriTAC-XRs targeting multiple tumor antigens resulted in a gradual build-up of active drug during the first week post dose and significantly lower Cmax/Cmin ratios than comparable constitutively active TriTACs. Modeling based on these PK data predicts that TriTAC-XR dosed intravenously will result in a slower build-up of active drug and smaller Cmax/Cmin ratios than TriTAC dosed intravenously or subcutaneously. Cytokine release and target cell depletion in cynomolgus monkeys were used to compare the therapeutic index of TriTAC-XR to TriTAC. A single dose of FLT3-targeting TriTAC-XR resulted in 100-fold protection in cytokine release and similar FLT3 expressing cell depletion when compared to an equivalent FLT3-targeting TriTAC. Similarly, repeat dosing of a TriTAC-XR targeting B cells resulted in complete B cell depletion with substantially lower cytokines than a comparable TriTAC. TriTAC-XR is expected to improve the safety of T cell engagers by reducing CRS and may increase clinical dosing convenience by enabling higher doses that will extend dosing intervals. Citation Format: Kathryn Strobel Kwant, Sony S. Rocha, Timothy Yu, Katrina Stephenson, Raphaela Rose Banzon, Sydney Vollhardt, Golzar Hemmati, Evan Callihan, Wade H. Aaron, Subramanian Thothathri, Jessica O'Rear, Eric Bragg, Willis Kwong, Hubert Situ, Avneel Hundal, Stephen Yu, Taggra Jackson, Kevin Carlin, Yinghua Xiao, Maria Rosalyn Dayao, Linh To, Nick Bergo, Kevin Wright, Richard Austin, Holger Wesche, Bryan Lemon, S. Jack Lin. TriTAC-XR: An extended-release T cell engager platform designed to minimize cytokine release syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2861.

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte–macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases., AhYoung, Eckard et al. show that the expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates the levels of histamine and serotonin in mast cell granules. This study reveals an important physiological function of NSP4 in mast cell-mediated vascular leakage in mice, establishing NSP4 as a potential therapeutic target for mast cell-dependent immune disorders.

Background Epithelial cell adhesion molecule (EpCAM) is highly expressed in many solid tumors. However, therapeutics targeting EpCAM have had limited clinical utility or failed in clinical development likely due to the expression of EpCAM in normal tissues. For example, clinical testing of solitomab, an EpCAM-targeting T cell engager, resulted in severe dose-limiting toxicities, including elevated liver transaminases, hyperbilirubinemia, and diarrhea. Designing an EpCAM-targeting T cell engager that is only active in the tumor would expand its therapeutic window and improve its safety profile. Methods Using a T cell engager prodrug platform named ProTriTAC that couples therapeutic half-life extension with functional masking, we have engineered HPN601, a protease-activated EpCAM-targeting T cell engager. HPN601 is a single polypeptide with three binding domains: anti-albumin for half-life extension, anti-CD3e for T cell engagement, and anti-EpCAM for tumor cell engagement. The anti-albumin domain contains a masking moiety and a protease-cleavable linker and keeps the molecule inert outside the tumor microenvironment. Activation by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potently active drug inside the tumor. This active drug has minimal activity outside of tumor because, without an albumin binding domain, it is rapidly cleared in circulation. Results A humanized rodent tumor model was used to simultaneously measure anti-tumor efficacy and clinically relevant toxicity endpoints. In this model, a surrogate molecule of HPN601 was safely administered at a dose ten-fold higher than the minimal efficacious dose required for durable tumor regression. Higher doses produced toxicities including elevated ALT/AST and bilirubin, body weight loss, and evidence of tissue damage by histopathology. In contrast, a constitutively active EpCAM-targeting T cell engager could only be dosed safely up to its minimal efficacious dose. The improved safety profile of HPN601 is further supported by a toxicokinetic study in non-human primates: compared to a constitutively active EpCAM-targeting T cell engager, HPN601 had significantly attenuated cytokine production, including IFN-g, IL-2, IL-6, and IL-10. Conclusions HPN601 is a conditionally active EpCAM-targeting T cell engager with a ten-fold improved therapeutic window compared to a constitutively active EpCAM-targeting T cell engager. An EpCAM-specific T cell engager with an improved safety profile could address unmet needs in many solid tumors and demonstrate the feasibility of using conditionally active T cell engagers to target more solid tumor antigens. Ethics Approval The study was reviewed and approved by Harpoon’s Institutional Animal Care and Use Committee.

Seizures are a disruption of normal brain activity present across a vast range of species and conditions. We introduce an organizing principle that leads to the first objective Taxonomy of Seizure Dynamics (TSD) based on bifurcation theory. The ‘dynamotype’ of a seizure is the dynamic composition that defines its observable characteristics, including how it starts, evolves and ends. Analyzing over 2000 focal-onset seizures from multiple centers, we find evidence of all 16 dynamotypes predicted in TSD. We demonstrate that patients’ dynamotypes evolve during their lifetime and display complex but systematic variations including hierarchy (certain types are more common), non-bijectivity (a patient may display multiple types) and pairing preference (multiple types may occur during one seizure). TSD provides a way to stratify patients in complement to present clinical classifications, a language to describe the most critical features of seizure dynamics, and a framework to guide future research focused on dynamical properties., eLife digest Epileptic seizures have been recognized for centuries. But it was only in the 1930s that it was realized that seizures are the result of out-of-control electrical activity in the brain. By placing electrodes on the scalp, doctors can identify when and where in the brain a seizure begins. But they cannot tell much about how the seizure behaves, that is, how it starts, stops or spreads to other areas. This makes it difficult to control and prevent seizures. It also helps explain why almost a third of patients with epilepsy continue to have seizures despite being on medication. Saggio, Crisp et al. have now approached this problem from a new angle using methods adapted from physics and engineering. In these fields, “dynamics research” has been used with great success to predict and control the behavior of complex systems like electrical power grids. Saggio, Crisp et al. reasoned that applying the same approach to the brain would reveal the dynamics of seizures and that such information could then be used to categorize seizures into groups with similar properties. This would in effect create for seizures what the periodic table is for the elements. Applying the dynamics research method to seizure data from more than a hundred patients from across the world revealed 16 types of seizure dynamics. These “dynamotypes” had distinct characteristics. Some were more common than others, and some tended to occur together. Individual patients showed different dynamotypes over time. By constructing a way to classify seizures based on the relationships between the dynamotypes, Saggio, Crisp et al. provide a new tool for clinicians and researchers studying epilepsy. Previous clinical tools have focused on the physical symptoms of a seizure (referred to as the phenotype) or its potential genetic causes (genotype). The current approach complements these tools by adding the dynamotype: how seizures start, spread and stop in the brain. This approach has the potential to lead to new branches of research and better understanding and treatment of seizures.

BackgroundCD3-targeted T cell engagers are potent anti-tumor therapies, but their development often requires management of cytokine release syndrome (CRS). Subcutaneous dosing is a promising strategy to reduce CRS, but its application is limited by its increased immunogenicity risks. Subcutaneous dosing is hypothesized to mitigate CRS by reducing the maximum drug concentration (Cmax) and preserve efficacy by maintaining the same minimum drug concentration (Cmin) as intravenous dosing. A T cell engager designed to be dosed intravenously but engineered to mimic the PK properties of subcutaneous dosing could alleviate CRS without increasing immunogenicity.MethodsTriTAC-XR molecules are engineered T cell engager prodrugs that become slowly activated in systemic circulation. This extended-release mechanism results in a slow build-up of circulating active drug, similar to subcutaneous dosing, and extends drug exposure to enable longer dosing intervals. The prodrug was engineered by adding a peptide mask and protease-cleavable linker to the N-terminus of a TriTAC, a constitutively active and half-life extended T cell engager. The mask binds to the anti-CD3ε domain and prevents T cell binding. Upon cleavage by systemic proteases, active T cell engager is released. Binding was assessed using ELISA on recombinant CD3ε protein and using flow cytometry on primary T cells. T cell engager function was assessed using T cell-dependent cellular cytotoxicity (TDCC) assays with resting human T cells. Safety and efficacy were modeled in non-human primates.ResultsTriTAC-XR had markedly reduced binding to recombinant CD3ε protein and to primary T cells as well as reduced potency in functional TDCC assays compared to its unmasked active drug. In cynomolgus monkeys, TriTAC-XR had significantly attenuated cytokine production while maintaining comparable pharmacodynamic effects as a non-masked active drug. The ratio of Cmax to Cmin for the active TriTAC-XR was significantly smaller than a non-masked control.ConclusionsTriTAC-XR is activated in a time released manner by systemic proteases to minimize differences between the Cmax and Cmin of systemic active drug. This mechanism is different from other protease-activated T cell engager prodrugs that are only activated by tumor-associated proteases. Compared to canonical T cell engagers, TriTAC-XR is expected to improve safety by reducing CRS and to provide convenience by extending dosing intervals.

Importance Many cancer subtypes, including KIT-mutant gastrointestinal stromal tumors (GISTs), are driven by activating mutations in tyrosine kinases and may initially respond to kinase inhibitors but frequently relapse owing to outgrowth of heterogeneous subclones with resistance mutations. KIT inhibitors commonly used to treat GIST (eg, imatinib and sunitinib) are inactive-state (type II) inhibitors. Objective To assess whether combining a type II KIT inhibitor with a conformation-complementary, active-state (type I) KIT inhibitor is associated with broad mutation coverage and global disease control. Design, setting, and participants A highly selective type I inhibitor of KIT, PLX9486, was tested in a 2-part phase 1b/2a trial. Part 1 (dose escalation) evaluated PLX9486 monotherapy in patients with solid tumors. Part 2e (extension) evaluated PLX9486-sunitinib combination in patients with GIST. Patients were enrolled from March 2015 through February 2019; data analysis was performed from May 2020 through July 2020. Interventions Participants received 250, 350, 500, and 1000 mg of PLX9486 alone (part 1) or 500 and 1000 mg of PLX9486 together with 25 or 37.5 mg of sunitinib (part 2e) continuously in 28-day dosing cycles until disease progression, treatment discontinuation, or withdrawal. Main outcomes and measures Pharmacokinetics, safety, and tumor responses were assessed. Clinical efficacy end points (progression-free survival and clinical benefit rate) were supplemented with longitudinal monitoring of KIT mutations in circulating tumor DNA. Results A total of 39 PLX9486-naive patients (median age, 57 years [range, 39-79 years]; 22 men [56.4%]; 35 [89.7%] with refractory GIST) were enrolled in the dose escalation and extension parts. The recommended phase 2 dose of PLX9486 was 1000 mg daily. At this dose, PLX9486 could be safely combined with 25 or 37.5 mg daily of sunitinib continuously. Patients with GIST who received PLX9486 at a dose of 500 mg or less, at the recommended phase 2 dose, and with sunitinib had median (95% CI) progression-free survivals of 1.74 (1.54-1.84), 5.75 (0.99-11.0), and 12.1 (1.34-NA) months and clinical benefit rates (95% CI) of 14% (0%-58%), 50% (21%-79%), and 80% (52%-96%), respectively. Conclusions and relevance In this phase 1b/2a nonrandomized clinical trial, type I and type II KIT inhibitors PLX9486 and sunitinib were safely coadministered at the recommended dose of both single agents in patients with refractory GIST. Results suggest that cotargeting 2 complementary conformational states of the same kinase was associated with clinical benefit with an acceptable safety profile. Trial registration ClinicalTrials.gov Identifier: NCT02401815.

T cell engagers have compelling clinical activity, but their use is limited by the availability of tumor targets with minimal normal tissue expression. Conditionally active T cell engagers designed to be active only in the tumor and to spare normal tissues could increase the number of addressable tumor targets. Here, we describe a T cell engager prodrug platform, named ProTriTAC, that is designed to be preferentially active in the tumor to reduce its on-target/off-tumor toxicity and improve the safety profile of T cell engagers targeting solid tumor antigens. ProTriTACs are based on the TriTAC platform with three binding domains on a single polypeptide: anti-albumin for half-life extension, anti-CD3 for T cell engagement, and anti-target antigen for tumor cell engagement. The anti-albumin domain, bearing a masking moiety and a protease-cleavable linker, can keep the prodrug inert with an extended half-life. Activation by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a short-lived active drug to direct T cell killing within the tumor. Multiple in vitro and in vivo approaches were used to demonstrate that ProTriTAC has an expanded therapeutic index (TI). In vitro binding and functional assays show that the prodrug has >1000x attenuated binding to primary human T cells and approximately 500x reduced T cell killing compared to its protease-activated active drug. Humanized rodent tumor xenograft models were used to simultaneously measure anti-tumor efficacy and clinically relevant toxicity endpoints with a ProTriTAC targeting the tumor antigen epithelial cell adhesion molecule (EpCAM). EpCAM ProTriTACs could be safely administered at a dose 10x higher than the minimal efficacious dose required for durable tumor regression. Higher doses of EpCAM ProTriTAC produced body weight loss, elevated ALT/AST and bilirubin, and evidence of tissue damage by histopathology. In contrast, a constitutively active EpCAM-targeting T cell engager could only be dosed safely up to its minimum efficacious dose. These data suggest that the conditionally active ProTriTAC confers a 10x TI expansion compared to the constitutively active T cell engager. Demonstration of TI expansion, in vitro and in vivo, for ProTriTACs targeting other tumor antigens will also be presented, demonstrating the robust applicability of the ProTriTAC platform across multiple tumor targets. Citation Format: S. Jack Lin, Sony S. Rocha, Kathryn Kwant, Maria Rosalyn Dayao, Tessie M. Ng, Raphaela Rose Banzon, Subramanian Thothathri, Wade Aaron, Evan Callihan, Golzar Hemmati, Kevin J. Wright, Manasi Barath, Yinghua Xiao, Linh To, Timothy Yu, Jessica O'Rear, Eric Bragg, Scott Gatto, Stephen Yu, Purbasa Patnaik, Avneel Hundal, Richard J. Austin, Bryan Lemon, Holger Wesche. ProTriTAC is a modular and robust T cell engager prodrug platform with therapeutic index expansion observed across multiple tumor targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 933.

Brackish coal seam water produced from the coal seam gas (CSG) industry is often treated by reverse osmosis to make the water suitable for beneficial reuse. As such, pre-treatment technologies are required in order to minimise membrane fouling. It was our hypothesis that coagulants may be able to remove problematic dissolved ions such as alkaline earth ions and soluble silicate species which are responsible for scaling of membranes and equipment. Consequently, this study evaluated the performance of aluminium chlorohydrate and aluminium sulphate coagulants for both simulated coal seam water and water collected from operating CSG wells. For simulated coal seam water samples the degree of dissolved silica removal was generally high (>85%) and promoted by increasing water salinity. Aluminium sulphate was better at removing silica ( c.f. 94–92%), barium ( c.f. 87–20%), strontium ( c.f. 66–15%) and magnesium ( c.f. 16–7.5%) and worse at removing calcium (44–65%) compared to aluminium chlorohydrate from simulated coal seam water, respectively. Variation in coal seam water composition of relatively concentrated solutions detrimentally impacted alkaline earth ion removal and in some instances reduced the extent of dissolved silica removal. When coal seam water samples for operating wells were studied, the amount of aluminium chlorohydrate required was significantly increased and the silica removal process inhibited (50% compared to 92% for simulated CS water). It was proposed that the presence of organic species in the real coal seam water played an important role in the mode by which the coagulants operated. Future studies should focus on understanding in more detail the influence of organic matter and influence of dissolved cations and anions upon coagulant behaviour.

Highlights - Iron coagulants effective for removal of dissolved Si, Ba, Ca and Sr from simulated coal seam water. - Salinity of CS water impacts the removal performance of silicate and alkaline earth ions. - Variable CS water compositions alter ion removal but not to extent of solution salinity changes. - Organic contaminants and particulates in real CS water inhibit coagulant performance. Abstract This study reports the novel application of iron based coagulants to remove dissolved species from coal seam water. Pre-treatment of coal seam (CS) water from coal seam gas operations is required to minimise fouling of downstream membranes. The behaviour of ferric chloride and ferric sulphate with both simulated and actual coal seam water was studied by jar tests. Dissolved silica was the major species eliminated by either coagulant with >80% removal for simulated coal seam water. Barium ions were highly removed, in contrast to magnesium. Salinity of the CS water sample influenced the coagulant ability to reduce dissolved silica which may be due to differences in floc structure and the cation demand of the solution. Varying the simulated CS water composition revealed that silica removal could be promoted albeit it was not clear this outcome was related to the presence of a particular species in solution. Use of real CS water resulted in inhibition of the silica removal mechanism which was ascribed to the presence of organic materials and turbidity which were not accounted for in simulated solutions. Residual iron levels also seemed to increase when real CS water was tested, and with ferric sulphate the issue of contamination of treated water with sulphate species arose, an issue which may impact downstream membrane fouling. As dissolved silicates and alkaline earth ions are of primary concern relating to scaling of water treatment equipment and membranes, a deeper understanding of coagulant performance is warranted.

Many risk genes for the development of Alzheimer’s disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis., Genetics implicate microglia in Alzheimer’s disease pathogenesis, but their roles remain unclear. Here, the authors find that microglial depletion in a mouse model of Alzheimer’s disease impairs plaque formation and that Aβ-induced changes in neuronal gene expression are microglia-mediated.

Constitutive modelling in geotechnical engineering and applied earth sciences is facing the challenge of assessing the long-term potential of both man-made and natural geohazards. This involves designing engineering operations for length scales and timescales well beyond those observable in the laboratory. In order to bridge the gap between scales, study rock behaviours well beyond their yield point and provide predictive capabilities to long term problems, constitutive models need to be equipped with multi-physical information that can be measured at laboratory time-scales and extrapolated beyond them. Enriching constitutive models with a sufficient level of details from the physical mechanisms taking place at the microstructure could provide such functionalities. In this work, a physics-based constitutive theory for sedimentary rocks is proposed combining the results of laboratory tests, theoretical analysis and numerical validation. The viscosity of the material is assumed to be a function of the temperature, pore-pressure and energy required to alter the inter-granular interfaces. The resulting flow law and corresponding stress equilibrium are coupled to the energy and mass conservation laws, constituting a closed system of equations. To solve this system, the theoretical framework is implemented using the Finite Element \redback{} simulator and its qualitative behaviour is analysed in monotonous and cyclic isotropic compression, as well as in direct shear for different loading rates. A series of numerical calibration tests is then performed against different types of rocks (sandstone, mudstone), saturating conditions (dry, wet), stress paths (triaxial, isotropic) and temperatures (from room temperature to over 800K). It concludes that the mechanical response of sedimentary porous rocks at strains usually achieved in laboratory testing is determined by the strength of the cementitious material bonding the grains. This strength is shown to be stress path dependent under the hypotheses made in this work and the interfaces are shown to obey a Kelvin-like interface law at the microscopic level.

As multiphysics geomechanical models get developed, their increasing complexity and number of parameters make it particularly difficult to calibrate against experimental data. In this contribution, we present a heuristic workflow to invert for parameters of a coupled Thermo-Hydro-Mechanical (THM) model in a way that helps the theoretical modellers refine their definition of the underlying elasto-visco-plastic model itself. We apply this workflow to the calibration of deviatoric and volumetric data for two sets of triaxial experiments on mudstone and sandstone. We show that beyond the calibration of well-defined parameters of the THM model, one or two scaling factors of the experimentally obtained yield surface and two more factors to capture the confinement and pore pressure dependency of the flow law lead to the satisfactory matching of series of experiments at varying confinements for two different rock types. Using this physical model, we also show that tracking the volumetric component of the mechanical power in the numerical simulations might allow reducing the number of experi- ments required to calibrate the model.

As energy operations face the challenge of reservoirs at ever-increasing depths, modelling the response of reservoir rocks at high pressure and high temperature (HPHT) conditions is a crucial step for successfully unlocking new resources. At these conditions, rocks can experience thermal and pressure sensitivity, as well as admit internal phase changes at the pore-solid interfaces that determine their macroscopic response to external loading. It is therefore expected that constitutive laws of Geomechanics should be enriched to accommodate such effects. In this work, a multi-physics constitutive theory for sedimentary rocks is proposed within the general framework of viscoplasticity. The viscosity of the material is assumed to be a function of the temperature, pore-pressure and energy required to alter the inter-granular interfaces. This energy is expressed through the chemical potentials of the phases involved during processes like chemical dissolution/precipitation of the interfaces or mechanical debonding. The resulting flow law and corresponding stress equilibrium are coupled to the energy and mass conservation laws, constituting a closed system of equations, which is solved using the Finite Element simulator REDBACK. A series of numerical tests for performance and calibration is then successfully performed against different types of reservoir rocks, confining pressures and temperatures (from room temperature to over 800K). We show that the mechanical response of sedimentary porous rocks at strains usually achieved in laboratory testing can be explained by accounting for the interface processes taking place at the cementitious material bonding the grains, a process that can also determine the brittle-to-ductile transition of sedimentary rocks.

As multiphysics geomechanical models get developed, their increasing complexity and number of parameters make it particularly difficult to calibrate against experimental data. In this contribution, we present a heuristic workflow to invert for parameters of a coupled Thermo-Hydro-Mechanical (THM) model in a way that helps the theoretical modellers refine their definition of the underlying elasto-visco-plastic model itself. We apply this workflow to the calibration of deviatoric and volumetric data for two sets of triaxial experiments on mudstone and sandstone. We show that beyond the calibration of well-defined parameters of the THM model, one or two scaling factors of the experimentally obtained yield surface and two more factors to capture the confinement and pore pressure dependency of the flow law lead to the satisfactory matching of series of experiments at varying confinements for two different rock types. Using this physical model, we also show that tracking the volumetric component of the mechanical power in the numerical simulations might allow reducing the number of experiments required to calibrate the model.

The purpose of the present study was to investigate the in vitro and in vivo activity of PLX9486, a tyrosine kinase inhibitor (TKI) targeting both primary KIT exon 9 and 11 and secondary exon 17 and 18 mutations in gastrointestinal stromal tumors (GISTs). Imatinib, a potent inhibitor of mutated KIT, has revolutionized the clinical management of advanced, metastatic GIST. However, secondary resistance develops mainly through acquired mutations in KIT exons 13/14 or exons 17/18. Second-line sunitinib potently inhibits KIT exon 13/14 mutants but is ineffective against exon 17 mutations. In our study, PLX9486 demonstrated in vitro nanomolar potency in inhibiting the growth and KIT phosphorylation of engineered BaF3 cells transformed with KIT exon 17 mutations (p.D816V) and with the double KIT exon 11/17 mutations (p.V560G/D816V). The in vivo efficacy of PLX9486 was evaluated using two imatinib-resistant GIST patient-derived xenograft (PDX) models. In UZLX-GIST9 (KIT: p.P577del;W557LfsX5;D820G), PLX9486 100 mg/kg/day resulted in significant inhibition of proliferation. Pharmacodynamic analysis showed a pronounced reduction in mitogen-activated protein kinase (MAPK) activation and other downstream effects of the KIT signaling pathway but no significant effect on KIT Y703 and Y719 phosphorylation. Similarly, in MRL-GIST1 (KIT: p.W557_K558del;Y823D) PLX9486 treatment led to significant tumor regression and strong inhibition of MAPK activation. Interestingly, the inhibitory effect on MAPK activation was evident even after a single dose of PLX9486. In conclusion, PLX9486 showed anti-tumor efficacy in patient-derived imatinib-resistant GIST xenograft models, mainly through inhibition of KIT signaling. These preclinical efficacy data encourage further testing of PLX9486 in the clinical setting.

The tolerability and ultimately efficacy of ADCs are limited by 2 major issues: (1) antigen expression that is too low on tumors, resulting in insufficient toxin delivery to the tumor, especially within the confines of the clinical MTD established by linker/payload-driven off-target toxicity and (2) too much antigen expression on normal healthy tissues, resulting in on-target but off-tumor toxicity. In this chapter, we will review strategies for making antibody prodrugs that have been or could be used to selectively deliver drug to a tumor compared to normal tissues. These technologies have the potential to lower on-target, off-tumor toxicities and enable better efficacy of ADCs due to better target selection and the delivery of higher concentrations of drug to tumors.

Oncogenic activation of BRAF fuels cancer growth by constitutively promoting RAS-independent mitogen-activated protein kinase (MAPK) pathway signalling. Accordingly, RAF inhibitors have brought substantially improved personalized treatment of metastatic melanoma. However, these targeted agents have also revealed an unexpected consequence: stimulated growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or paradoxically activate the MAPK pathway, depending whether activation is by BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. Here we have identified next-generation RAF inhibitors (dubbed 'paradox breakers') that suppress mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In cells that express the same HRAS mutation prevalent in squamous tumours from patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated in vitro and in vivo growth and induced expression of MAPK pathway response genes; by contrast the paradox breakers PLX7904 and PLX8394 had no effect. Paradox breakers also overcame several known mechanisms of resistance to first-generation RAF inhibitors. Dissociating MAPK pathway inhibition from paradoxical activation might yield both improved safety and more durable efficacy than first-generation RAF inhibitors, a concept currently undergoing human clinical evaluation with PLX8394.