A Cell Surface-Binding Antibody Atlas Nominates a MUC18-Directed Antibody-Drug Conjugate for Targeting Melanoma.

Recent advances in targeted therapy and immunotherapy have substantially improved the treatment of melanoma. However, therapeutic strategies are still needed for unresponsive or treatment-relapsed patients with melanoma. To discover antibody-drug conjugate (ADC)-tractable cell surface targets for melanoma, we developed an atlas of melanoma cell surface-binding antibodies (pAb) using a proteome-scale antibody array platform. Target identification of pAbs led to development of melanoma cell killing ADCs against LGR6, TRPM1, ASAP1, and MUC18, among others. MUC18 was overexpressed in both tumor cells and tumor-infiltrating blood vessels across major melanoma subtypes, making it a potential dual-compartment and universal melanoma therapeutic target. AMT-253, an MUC18-directed ADC based on topoisomerase I inhibitor exatecan and a self-immolative T moiety, had a higher therapeutic index compared with its microtubule inhibitor-based counterpart and favorable pharmacokinetics and tolerability in monkeys. AMT-253 exhibited MUC18-specific cytotoxicity through DNA damage and apoptosis and a strong bystander killing effect, leading to potent antitumor activities against melanoma cell line and patient-derived xenograft models. Tumor vasculature targeting by a mouse MUC18-specific antibody-T1000-exatecan conjugate inhibited tumor growth in human melanoma xenografts. Combination therapy of AMT-253 with an antiangiogenic agent generated higher efficacy than single agent in a mucosal melanoma model. Beyond melanoma, AMT-253 was also efficacious in a wide range of MUC18-expressing solid tumors. Efficient target/antibody discovery in combination with the T moiety-exatecan linker-payload exemplified here may facilitate discovery of new ADC to improve cancer treatment. SIGNIFICANCE: Discovery of melanoma-targeting antibodies using a proteome-scale array and use of a cutting-edge linker-payload system led to development of a MUC18-targeting antibody-exatecan conjugate with clinical potential for treating major melanoma subtypes.

AMT-562, a Novel HER3-targeting Antibody-Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors.

HER3 is a unique member of the EGFR family of tyrosine kinases, which is broadly expressed in several cancers, including breast, lung, pancreatic, colorectal, gastric, prostate, and bladder cancers and is often associated with poor patient outcomes and therapeutic resistance. U3-1402/Patritumab-GGFG-DXd is the first successful HER3-targeting antibody-drug conjugate (ADC) with clinical efficacy in non-small cell lung cancer. However, over 60% of patients are nonresponsive to U3-1402 due to low target expression levels and responses tend to be in patients with higher target expression levels. U3-1402 is also ineffective in more challenging tumor types such as colorectal cancer. AMT-562 was generated by a novel anti-HER3 antibody Ab562 and a modified self-immolative PABC spacer (T800) to conjugate exatecan. Exatecan showed higher cytotoxic potency than its derivative DXd. Ab562 was selected because of its moderate affinity for minimizing potential toxicity and improving tumor penetration purposes. Both alone or in combination therapies, AMT-562 showed potent and durable antitumor response in low HER3 expression xenograft and heterogeneous patient-derived xenograft/organoid models, including digestive system and lung tumors representing of unmet needs. Combination therapies pairing AMT-562 with therapeutic antibodies, inhibitors of CHEK1, KRAS, and tyrosine kinase inhibitor showed higher synergistic efficacy than Patritumab-GGFG-DXd. Pharmacokinetic and safety profiles of AMT-562 were favorable and the highest dose lacking severe toxicity was 30 mg/kg in cynomolgus monkeys. AMT-562 has potential to be a superior HER3-targeting ADC with a higher therapeutic window that can overcome resistance to generate higher percentage and more durable responses in U3-1402-insensitive tumors.

Antibody-Exatecan Conjugates with a Novel Self-immolative Moiety Overcome Resistance in Colon and Lung Cancer.

Antibody-drug conjugates (ADC) using DNA topoisomerase I inhibitor DXd/SN-38 have transformed cancer treatment, yet more effective ADCs are needed for overcoming resistance. We have designed an ADC class using a novel self-immolative T moiety for traceless conjugation and release of exatecan, a more potent topoisomerase I inhibitor with less sensitivity to multidrug resistance (MDR). Characterized by enhanced therapeutic indices, higher stability, and improved intratumoral pharmacodynamic response, antibody-T moiety-exatecan conjugates targeting HER2, HER3, and TROP2 overcome the intrinsic or treatment resistance of equivalent DXd/SN-38 ADCs in low-target-expression, large, and MDR+ tumors. T moiety-exatecan ADCs display durable antitumor activity in patient-derived xenograft and organoid models representative of unmet clinical needs, including EGFR ex19del/T790M/C797S triple-mutation lung cancer and BRAF/KRAS-TP53 double-mutant colon cancer, and show synergy with PARP/ATR inhibitor and anti-PD-1 treatment. High tolerability of the T moiety-exatecan ADC class in nonhuman primates supports its potential to expand the responding patient population and tumor types beyond current ADCs. SIGNIFICANCE: ADCs combining a novel self-immolative moiety and topoisomerase I inhibitor exatecan as payload show deep and durable response in low-target-expressing and MDR+ tumors resistant to DXd/SN-38 ADCs without increasing toxicity. This new class of ADCs has the potential to benefit an additional patient population beyond current options. See related commentary by Gupta et al., p. 817. This article is highlighted in the In This Issue feature, p. 799.

Identification of Human UMP/CMP Kinase 1 as Doxorubicin Binding Target Using Protein Microarray.

Doxorubicin (DOX) is a leading anthracycline drug with exceptional efficacy; however, little is known about the molecular mechanisms of its side effects, which include heart muscle damage, noncancerous cell death, and drug resistance. A total of 17,950 human proteins expressed in HEK293 cells were screened and yielded 14 hits. Competitive and binding experiments further verified the binding of DOX to UMP/CMP kinase 1 (CMPK1), and microscale thermophoresis showed that DOX binds to CMPK1 with a Kd of 1216 nM. In addition, we observed that the binding of DOX to CMPK1 activated the phosphorylation of CMP, dCMP, and UMP. A significant activation was observed at the concentration of 30 µM DOX and reached plateau at the concentration of DOX 30 µM, 150 µM, and 100 µM, respectively. DOX would add up stimulation of CMPK1 by DTT and overcome inhibition of CMPK1 by NaF, EDTA. In summary, we showed that DOX might bind to the nonactive site of CMPK1 and regulate its activity with magnesium.

Synthetic B- and T-cell epitope peptides of porcine reproductive and respiratory syndrome virus with Gp96 as adjuvant induced humoral and cell-mediated immunity.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vaccines and attenuated live vaccines, are available but fail to provide sustainable protection, especially against genetically heterologous strains. Thus several approaches have been used to develop more effective PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22-355 aa of Gp96 (Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated PRRSV-specific humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specific cellular immunity induced by TCE-peptides. Moreover, higher levels of IL-12 and TNF-α and lower levels of IL-4 and IL-10 were observed in the serum of Gp96N-vaccinated piglets compared to piglets immunized with no Gp96N, displaying a predominant Th1 type of immune response induced by Gp96N. Following challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptides and Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infection. These data provide important bases for the development of PRRSV epitope-based synthetic peptide vaccines combined with Gp96N as attractive immunomodulators in swine.

Development of a recombinant N-Gp5c fusion protein-based ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus.

The recent dramatic increase in reported cases of porcine reproductive and respiratory syndrome (PRRS) in pig farms is a potential threat to the global swine industry, and thus, detecting PRRS virus (PRRSV) in pig herds is essential to help control the spread of PRRS. IDEXX HerdChek™ PRRS, a commercially available indirect enzyme-linked immunosorbent assay (iELISA), is the industry standard for detection of antibodies against PRRSV. In the present study, an effective iELISA for detection of PRRSV antibodies was developed using a recombinant fusion protein N-Gp5c (rN5c, a combination of the nucleocapsid protein and the C-terminal 78 aa of Gp5) produced in Escherichia coli. This assay was validated by comparison with an immunofluorescent assay and IDEXX-ELISA. The diagnostic specificity, sensitivity, and accuracy of the rN5c-iELISA method were 94.8, 95.6, and 95.1%, respectively. Cross-reactivity assays demonstrated that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 13 and 22%, respectively. The rN5c-iELISA is simpler to produce and perform, time-saving, and suitable for large scale surveys of PRRSV infection at lower cost.

Gp96 enhances the immunogenicity of subunit vaccine of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry worldwide. Currently available commercial vaccines provide limited protection due to delayed and weak cell-mediated immunity and neutralizing antibody production, thus the immunomodulators should be considered in order to improve the efficacy of PRRSV vaccines. Heat shock protein gp96 may be used as a modulator to enhance both innate and adaptive immune responses. In the present study, two multi-epitope subunit vaccines, named as Cp1 and Cp2, were designed based on the conserved B cell epitopes of viral proteins with the N-terminal 22-370 amino acids (aa) of porcine gp96 (Gp96N) chosen as the adjuvant. Immune responses elicited by the different combinations of Cp1/Cp2 and Gp96N were examined in mice and piglets. The results indicated that the group of Cp1/Cp2-Gp96N (CG) combination induced 3-4-fold higher titers of Cp1/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups which received Cp1/Cp2 immunization alone or with Freund's adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from vaccinated mice or piglets. The production of IFN-γ in mice splenocytes, TNF-α, IFN-γ, and IL-12 in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4 was reduced by half and IL-10 was decreased to an undetectable level. These results suggest that the porcine Gp96N could effectively enhance the innate and adaptive immune responses of Cp1/Cp2 with a Th1-type bias. Therefore, the multi-epitope subunit vaccine Cp1/Cp2 co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.

Identification and characterization of three novel nuclear export signals in the influenza A virus nucleoprotein.

The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP--NES1, NES2, and NES3--were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.

Cyclophilin E functions as a negative regulator to influenza virus replication by impairing the formation of the viral ribonucleoprotein complex.

BACKGROUND: The nucleoprotein (NP) of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE), a member of the peptidyl-propyl cis-trans isomerase (PPIase) family, was found to bind to NP and inhibit viral replication and transcription. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, CypE was found to interact with NP but not with the other components of the viral ribonucleoprotein complex (VRNP): PB1, PB2, and PA. Mutagenesis data revealed that the CypE domain comprised of residues 137-186 is responsible for its binding to NP. Functional analysis results indicated that CypE is a negative regulator in the influenza virus life cycle. Furthermore, knock-down of CypE resulted in increased levels of three types of viral RNA, suggesting that CypE negatively affects viral replication and transcription. Moreover, up-regulation of CypE inhibited the activity of influenza viral polymerase. We determined that the molecular mechanism by which CypE negatively regulates influenza virus replication and transcription is by interfering with NP self-association and the NP-PB1 and NP-PB2 interactions. CONCLUSIONS/SIGNIFICANCE: CypE is a host restriction factor that inhibits the functions of NP, as well as viral replication and transcription, by impairing the formation of the vRNP. The data presented here will help us to better understand the molecular mechanisms of host restriction barriers, host adaptation, and tissue tropism of influenza A virus.

[Overview of Gp96 mediated immunity].

As a member of the HSP90 family, heat shock protein (HSP) Gp96 is one of the most abundant proteins in the endoplasmic reticulum (ER), which displayed important molecular chaperones function in cells. Gp96 can stimulate the production of cytokines by activating the antigen presentation cells (such as dendritic cell, et al) in innate immunity. It is capable of eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity. Gp96 is also an ideal adjuvant in many recent researches. Here, we review the progress that addresses the role of biological characteristics, immunogenic mechanism that may be involved in the induction of anti-infection immune response and antitumor immunity, which may guide the new vaccine strategies with the knowledge of Gp96-antigen complexes.

Subcellular localization and topology of porcine reproductive and respiratory syndrome virus E protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 70 or 73 amino acids (aa), termed E protein. The function and biochemical information of the E protein are currently not clear. In the present investigation, it was shown that the E protein was mainly located in the endoplasmic reticulum (ER) and Golgi complex in MARC-145 cells. Deletion studies identified the N-terminal 15 residues as an ER localization sequence of the E protein, besides two other localization sequences within positions 23-50 and 50-73, and the N-myristoylation site significantly affected the subcellular localization of the N-terminal 15 residues. The membrane association assay demonstrated that the E protein was an integral membrane protein embedded in the phospholipid bilayer. However, neither the N-myristoylation site nor the hydrophilic C-terminal domain was essential to the membrane association of the E protein. The topology analysis revealed that this protein had N-terminus oriented toward the cytoplasm and C-terminus toward the ER lumen. Finally, immunofluorescence assay indicated that the E protein colocalized with GP2, GP3, GP4 and M protein in cotransfected cells, but not N protein.