Combined therapy of dabrafenib and an anti-HER2 antibody-drug conjugate for advanced BRAF-mutant melanoma.

BACKGROUND: Melanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma. METHODS: We evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy. RESULTS: Our results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway. CONCLUSION: These findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Engineering hydrophobicity and manufacturability for optimized biparatopic antibody-drug conjugates targeting c-MET.

Optimal combinations of paratopes assembled into a biparatopic antibody have the capacity to mediate high-grade target cross-linking on cell membranes, leading to degradation of the target, as well as antibody and payload delivery in the case of an antibody-drug conjugate (ADC). In the work presented here, molecular docking suggested a suitable paratope combination targeting c-MET, but hydrophobic patches in essential binding regions of one moiety necessitated engineering. In addition to rational design of HCDR2 and HCDR3 mutations, site-specific spiking libraries were generated and screened in yeast and mammalian surface display approaches. Comparative analyses revealed similar positions amendable for hydrophobicity reduction, with a broad combinatorial diversity obtained from library outputs. Optimized variants showed high stability, strongly reduced hydrophobicity, retained affinities supporting the desired functionality and enhanced producibility. The resulting biparatopic anti-c-MET ADCs were comparably active on c-MET expressing tumor cell lines as REGN5093 exatecan DAR6 ADC. Structural molecular modeling of paratope combinations for preferential inter-target binding combined with protein engineering for manufacturability yielded deep insights into the capabilities of rational and library approaches. The methodologies of in silico hydrophobicity identification and sequence optimization could serve as a blueprint for rapid development of optimal biparatopic ADCs targeting further tumor-associated antigens in the future.

Genetic code expansion in E. coli enables production of a functional 'ready-to-click' T cell receptor-specific scFv.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Light Chain Q166C Mutation Permits One-step Site Specific Conjugation on Monoclonal Antibodies.

Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in in vitro cell migration and in vivo mouse xenograft models.

Pan-cancer gene expression analysis of tissue microarray using EdgeSeq oncology biomarker panel and a cross-comparison with HER2 and HER3 immunohistochemical analysis.

Molecular and protein biomarker profiling are key to oncology drug development. Antibody-drug conjugates (ADCs) directly deliver chemotherapeutic agents into tumor cells based on unique cancer cell biomarkers. A pan-cancer tissue microarray (TMA) data set and gene panel were validated and gene signature analyses were conducted on normal and cancer tissues to refine selection of ADC targets. Correlation of mRNA and protein levels, and human epidermal growth factor receptor (HER) expression patterns were assessed. An EdgeSeq biomarker panel (2862 genes) was used across 8531 samples (23 solid cancer types/subtypes; 16 normal tissues) with an established TMA data set, and immune cell and cell cycle gene signatures were analyzed. Discriminating gene expression signatures were defined based on pathological classification of cancer subtypes. Correlative analyses of HER2 and HER3 mRNA (EdgeSeq) and protein expression (immunohistochemistry [IHC]) were performed and compared with publicly available data (The Cancer Genome Atlas [TCGA]; Cancer Cell Line Encyclopedia [CCLE]). Gene expression patterns among cancer types in the TMA (EdgeSeq) and TCGA (RNA-seq) were similar. EdgeSeq gene signature analyses aligned with the majority of pathological cancer types/subtypes and identified cancer-specific gene expression patterns. TMA IHC H-scores for HER3 varied across cancer types/subtypes. In a few cancer types, HER3 mRNA and protein expression did not align, including lower liver hepatocellular carcinoma IHC H-score, compared with mRNA. Although all TNBC and ovarian cancer subtypes expressed mRNA, some had lower protein expression. This was seen in TMA and TCGA data sets, but not in CCLE. The EdgeSeq TMA data set can expand upon current biomarker data by including cancers not currently in TCGA. The primary analysis of EdgeSeq and IHC comparison suggested a unique protein-level regulation of HER3 in some tumor subtypes and highlights the importance of investigating protein levels of ADC targets in both tumor and normal tissues.

Discerning subsets of breast cancer with very low and absent HER2 protein expression.

Breast cancers are currently eligible for treatment with anti-HER2 therapies if they exhibit amplification of the gene ERBB2 and overexpression of its protein product HER2. Recently, breast cancers with low HER2 expression have shown response to novel anti-HER2 antibody-drug conjugates, and the lower end of "low-HER2" tumors has not yet been clinically delineated. The historically binary approach to HER2 scoring will need to evolve and reporting of HER2 status may require refinement to better stratify low-HER2 statuses. We performed a quality review of HER2 immunohistochemical (IHC) scoring of breast carcinomas with low HER2 expression (71 core biopsies and 51 excisions). We also investigated the feasibility of discerning cases with total lack of HER2 expression from those cases with "very low" HER2 expression that did not meet current criteria for a HER2(1+) score. Rescoring HER2 achieved substantial agreement when performed at 200×, and near-perfect agreement at 400× magnification. Examination under 400× magnification led to recognition of more cases with HER2 expression. Less than 10% of cases showed complete lack of HER2 protein expression by IHC. Cases with "very low" expression were readily identified, and such a category would be feasible to implement in pathologist workflow.

Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

Repeated Systemic Dosing of Adeno-Associated Virus Vectors in Immunocompetent Mice After Blockade of T Cell Costimulatory Pathways.

Neutralizing antibodies (NAbs) strongly limit adeno-associated virus (AAV) vector transduction and repeated administration. Previous studies have shown that NAbs induced by AAVs are associated with T and B cell activation and that the B7/CD28 and CD40/CD40L costimulation signaling pathways are involved. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD40 are vital molecules that participate in the costimulatory pathway. In this study, we evaluated CTLA4-Ig and CD40-Ig immunosuppreve efficacies through AAV and investigated their effects on the feasibility for multiple systemic administrations of AAV vectors. The results showed that a single administration of AAV vector carrying either CTLA4-Ig alone or with CD40-Ig could greatly reduce the level of NAbs. An AAV serotype-specific immune tolerance could be successfully established, which enabled repeated, that is, second and third, systemic administration of AAV vectors in the same mice. A combination of CTLA4-Ig and CD40-Ig delivered via AAV vectors significantly inhibited T and B cell activations without affecting the immune response to the total immunoglobulin G production and cytokines. Interestingly, exogenous gene expression significantly improved after multiple administrations of AAV vector in vivo. Our study generates a reliable and effective method for repeated dosing of AAV vectors that is needed on gene therapy.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

cDNA Display-Mediated Immuno-PCR (cD-IPCR): An Ultrasensitive Immunoassay for Biomolecular Detection.

Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

BACKGROUND & AIMS: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. METHODS: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. RESULTS: AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. CONCLUSIONS: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Biological evaluation of new TEM1 targeting recombinant antibodies for radioimmunotherapy: In vitro, in vivo and in silico studies.

The tumour endothelial marker 1 (TEM1/endosialin/CD248) is a receptor overexpressed in several human solid tumours and silenced in normal adult tissues, representing a suitable and potentially safe target for radioimmunotherapy of sarcoma. To develop new tools with improved TEM1 targeting properties, a new panel of antibody fragments was for the first time evaluated preclinically following 125I radiolabelling. The antibody fragment 1C1m-Fc, with the highest human/murine TEM1 binding affinity, was extensively characterized in vitro and in vivo in a Ewing's sarcoma human xenograft mouse model. In silico studies were also performed to elucidate the influence of a single amino acid mutation in the complementarity-determining region (CDR3) of the heavy chain, upon affinity maturation of the parental clone 1C1-Fc. From this study, 1C1m-Fc emerged as a promising candidate for the development of TEM1-targeted radioimmunoconjugates, namely to be further explored for theranostic applications with other suitable medical radionuclides.

Multicentric MFI30 study: Standardization of flow cytometry analysis of CD30 expression in non-Hodgkin lymphoma.

CD30 transmembrane receptor, a member of the tumor necrosis factor receptor family, is expressed in different lymphomas. Brentuximab vedotin (BV), a CD30 monoclonal antibody (Ab)-drug conjugate, is effective in CD30-positive lymphomas. However, the response to BV is not always correlated to CD30 expression detected by immunohistochemistry (IHC). The objectives of this study were to standardize and evaluate CD30 intensity by flow cytometry (FCM) in non-Hodgkin's lymphomas. Twelve centers analyzed 161 cases on standardized cytometers using normalized median fluorescence intensity (nMFI30) of three different Abs, of which one clone can recognize the same epitope as BV. FCM distinguished four groups of cases: negative group (n = 110) which showed no expression with the three clones; high positive group (n = 13) which gave nMFI30 > 5% with all tested clones; dim positive group (n = 17) which showed nMFI30 > 1% with all tested clones and <5% for at least one; discordant group (n = 21) with positive and negative expression of the different clones. In consistency with the literature, CD30 was positive in all anaplastic large cell lymphomas, in some diffuse large B-cell lymphomas (DLBCL), and in other rare lymphomas. FCM results were concordant with those of IHC in 77% of cases. Discrepancies could be explained by clones-related differences, microenvironment, or intracytoplasmic staining. Interestingly, FCM was more sensitive than IHC in 11% of cases, especially in DLBCL. Multicenter standardized FCM of specific CD30 could improve case detection and extend the treatment of BV to various CD30-positive lymphomas.

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities.

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

Phase I study of bintrafusp alfa, a bifunctional fusion protein targeting TGF-ß and PD-L1, in patients with pretreated biliary tract cancer.

BACKGROUND: Patients with biliary tract cancer (BTC) have poor prognosis with few treatment options. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor (TGF)-ßRII receptor (a TGF-ß 'trap') fused to a human IgG1 antibody blocking programmed death ligand 1 (PD-L1), has shown clinical efficacy in multiple solid tumors. METHODS: In this phase I, open-label trial expansion cohort, Asian patients with BTC whose disease progressed after first-line chemotherapy received bintrafusp alfa 1200 mg every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint is safety/tolerability, while the secondary endpoints include best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: As of August 24, 2018, 30 patients have received bintrafusp alfa for a median of 8.9 (IQR 5.7-32.1) weeks; 3 patients remained on treatment for >59.7 weeks. Nineteen (63%) patients experienced treatment-related adverse events (TRAEs), most commonly rash (17%), maculopapular rash and fever (13% each), and increased lipase (10%). Eleven (37%) patients had grade ≥3 TRAEs; three patients had grade 5 events (septic shock due to bacteremia, n=1; interstitial lung disease (reported term: interstitial pneumonitis), n=2). The objective response rate was 20% (95% CI 8 to 39) per independent review committee (IRC), with five of six responses ongoing (12.5+ to 14.5+ months) at data cut-off. Two additional patients with durable stable disease had a partial response per investigator. Median progression-free survival assessed by IRC and overall survival were 2.5 months (95% CI 1.3 to 5.6) and 12.7 months (95% CI 6.7 to 15.7), respectively. Clinical activity was observed irrespective of PD-L1 expression and microsatellite instability-high status. CONCLUSIONS: Bintrafusp alfa had clinical activity in Asian patients with pretreated BTC, with durable responses. Based on these results, bintrafusp alfa is under further investigation in patients with BTC (NCT03833661 and NCT04066491). TRIAL REGISTRATION NUMBER: NCT02699515.

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Advances in Antibody-Drug Conjugate Design: Current Clinical Landscape and Future Innovations.

The targeted delivery of potent cytotoxic molecules into cancer cells is considered a promising anticancer strategy. The design of clinically effective antibody-drug conjugates (ADCs), in which biologically active drugs are coupled through chemical linkers to monoclonal antibodies, has presented challenges for pharmaceutical researchers. After 30 years of intensive research and development activities, only seven ADCs have been approved for clinical use; two have received fast-track designation and two breakthrough therapy designation from the Food and Drug Administration. There is continued interest in the field, as documented by the growing number of candidates in clinical development. This review aims to summarize the most recent innovations that have been applied to the design of ADCs undergoing early- and late-stage clinical trials. Discovery and rational optimization of new payloads, chemical linkers, and antibody formats have improved the therapeutic index of next-generation ADCs, ultimately resulting in improved clinical benefit for the patients.