Gene fusions generated by chromosomal rearrangements function as oncogenic drivers in human cancers. We previously showed that EWSR1-ETS oncofusions of Ewing sarcoma directly induce surface expression of IL1 receptor accessory protein (IL1RAP), which along with limited expression in healthy tissues except in the placenta nominate IL1RAP as a promising Ewing sarcoma immunotherapy target. We therefore engineered antibody-drug conjugates (ADC) with different cytotoxic payloads to target IL1RAP. ADCs potently blocked tumor growth and induced durable regression of Ewing sarcoma xenografts in mice and diminished metastatic dissemination in vivo. Moreover, we show that other oncofusions also induce IL1RAP expression in diverse cancers, including NPM-ALK in anaplastic large cell lymphoma and ETV6-NTRK3 in multiple tumor types. IL1RAP expression rendered these malignancies similarly vulnerable to IL1RAP-targeting ADCs, which effectively blocked the growth of ALCL xenografts and syngeneic ETV6-NTRK3+ sarcomas. Lack of detectable normal tissue toxicity, including in nonhuman primates, supports the further clinical translation of IL1RAP-targeting ADCs. SIGNIFICANCE:The IL1RAP surface protein is induced by multiple oncogenic fusions, including EWSR1-ETS, NPM-ALK, and EN in distinct malignancies, rendering these cancers vulnerable to anti-IL1RAP ADCs. We demonstrate that IL1RAP-targeting ADCs have potent efficacy in both primary and metastatic diseases and are well tolerated in nonhuman primates, warranting their further clinical translation.

NK and T cells are key effectors that eliminate cancer cells, but upregulation of the inhibitory receptor NKG2A on these cells attenuates antitumor immune responses. To counteract NKG2A inhibitory signaling, we identified two specific fully human monoclonal anti-NKG2A antibodies that block HLA-E ligand binding. These antibodies activated NK cells and enhanced antibody-dependent cellular cytotoxicity of tumor-targeting IgG1s both in vitro and in vivo. Bispecific engagers (BiNKs), generated by fusing NKG2A antibodies with tumor targeting binders, promoted immune synapse formation and directed cytotoxicity of NK and CD8+ T cells toward cancer cells. In a human PBMC-engrafted NSG mouse xenograft lung cancer model, an anti-HER2 × anti-NKG2A BiNK markedly inhibited tumor growth as a monotherapy or in combination with pertuzumab. Cell depletion studies revealed that the BiNK enhanced antitumor activity of both NK and T cells. NKG2A blockade with potent and specific, fully human antibodies and BiNKs show promise for further development as cancer immunotherapeutics.

Human immunodeficiency virus (HIV) infection remains a global health threat. Although antiretroviral therapy (ART) has significantly transformed HIV into a manageable chronic disease, emergence of drug resistance to current ART is a continuing concern. Broadly neutralizing antibodies, as well as reagents containing both a soluble CD4 mimetic and an HIV co-receptor inhibitor, such as CD4-CD4i antibodies, are promising strategies for the prevention and treatment of HIV infection. We previously developed a CD4 D1mimetic (mD1.22) with enhanced neutralization potency, surpassing that of the clinically validated sCD4-D1D2 mimetic. We also previously identified a novel CD4i antibody (X5) that targets the conserved coreceptor binding site on the gp120 core and recognizes an epitope partially overlapping with 17b monoclonal antibody binding site. X5 binding to gp120 was augmented by CD4 and modestly enhanced by CCR5. To leverage these favorable interactions, we designed and optimized sCD4-X5 bispecific antibodies by computational structure-aided modification of antibody size and fusion linkers between the two binding moieties. The bispecific D1X5, with a (G4S)7 long linker between D1 and X5 (IgG1-LL D1X5), exhibited broad neutralization against 11 diverse HIV subtypes across B, C, G clades and AC, BC recombinants. The TZM-bl cell neutralization assay showed IgG1-LL D1X5 neutralization geometric mean IC50 and IC80 are 0.6 ug/mL and 3.4 ug/mL respectively, which are within the range of potent bnAbs. This work has identified a novel single domain soluble CD4 based CD4-CD4i bispecific antibody with broad HIV-1 neutralization.

Background: Macrophages play an important role in eliminating diseased and damaged cells through programmed cell death. Signal regulatory protein alpha (SIRPα) is a crucial immune checkpoint primarily expressed on myeloid cells and macrophages. It initiates a ‘do not eat me’ signal when engaged with CD47, which is typically expressed at elevated levels on multiple solid tumors. The phospholipase A2 Group 7 (PLA2G7), which is mainly secreted by macrophages, interacts with oxidized low-density lipoprotein (oxLDL) and associates with several vascular diseases and cancers. Methods: To identify potent fully human monoclonal antibodies (mAbs) against human SIRPα and PLA2G7, we conducted bio-panning of phage antibody libraries. Results: We isolated one human Fab (1B3) and VH (1A3) for SIRPα, as well as one human Fab (1H8) and one VH (1A9) for PLA2G7; the 1B3 Fab and 1A3 VH are competitively bound to SIRPα, interfering with CD47 binding. The 1B3 IgG and 1A3 VH-Fc augmented macrophage-mediated phagocytic activity when combined with the anti-EGFR antibody, cetuximab. The anti-PLA2G7 antibodies exhibited high specificity for the PLA2G7 antigen and effectively blocked the PLA2G7 enzymatic activity with half-maximal inhibitory concentrations (IC50) in the single-digit nanomolar range. Additionally, 1H8 IgG and its derivative bispecific antibody exhibited the ability to block PLA2G7-mediated tumor cell migration. Conclusions: Our anti-SIRPα mAbs are expected to serve as potent and fully human immune checkpoint inhibitors of SIRPα, enhancing the antitumor responses of SIRPα-positive immune cells. Moreover, our anti-PLA2G7 mAbs represent promising fully human PLA2G7 enzymatic blockade antibodies with the potential to enhance both anti-tumor and anti-aging responses. Anti-SIRPα and PLA2G7 mAbs can modulate macrophage phagocytic activity and inflammatory responses against tumors.

While some studies have implicated the histone 3 lysine 4 demethylase KDM5A in tumorigenesis and promotion of metastasis, KDM5A has also been shown to boost immunotherapy responses in melanoma. The distinct functional role of KDM5A in the context of immunotherapy and the underlying mechanisms remain largely unknown. Here, we report that higher KDM5A expression strongly correlates with higher MHC-I expression, CD8+ T cell infiltration, and prolonged survival in colorectal cancer and gastric cancer patients receiving anti– PD-1 therapy. KDM5A promotes MHC-I-mediated antigen presentation and CD8+ T cell-mediated tumor killing across multiple murine cancer models. Mechanistically, KDM5A upregulates MHC-I expression through the SOCS1/IFN-γ/STAT1 signaling and improves tumor cell-intrinsic antigen presentation capacity by inhibiting lysosomal proteases in a demethylase-dependent manner. Moreover, KDM5A directly represses lysosomal cathepsins in dendritic cells and promotes the cross-priming activity. Through a high-throughput chemical compound library-based screen, we identified Clomiphene, an FDA-approved small molecule, which significantly elevates KDM5A expression, and enhances antitumor immunity during anti–PD-1 immunotherapy in mouse models of melanoma and colon cancer. Our findings that KDM5A functions in MHC-I-mediated immune activation during anti–PD-1 therapy present an opportunity for developing KDM5A-enhancing therapies to increase tumor immunogenicity, sensitize solid tumors to immunotherapy and exclude immune evasion. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 21877067 Tsinghua-Peking Centre for Life Science Roche Postdoc Fellowship Program

The rapid evolution of the viral genome has led to the continual generation of new variants of SARS- CoV-2. Developing antibody drugs with broad- spectrum and high efficiency is a long- term task. It is promising but challenging to develop therapeutic neutralizing antibodies (nAbs) through in vitro evolution based on antigen-antibody binding interactions. From an early B cell antibody repertoire, we isolated antibody 8G3 that retains its nonregressive neutralizing activity against Omicron BA.1 and various other strains in vitro. 8G3 protected ACE2 transgenic mice from BA.1 and WA1/2020 virus infection without adverse clinical manifestations and completely cleared viral load in the lungs. Similar to most IGHV3-53 antibodies, the binding sites of 8G3 and ACE2 largely overlap, enabling competition with ACE2 for binding to RBD. By comprehensively considering the binding free energy changes of the antigen-antibody complexes, the biological environment of their interactions, and the evolutionary direction of the antibodies, we were able to select 50 mutants. Among them, 11 were validated by experiments showing better neutralizing activities. Further, a combination of four mutations were identified in 8G3 that increased its neutralization potency against JN.1, the latest Omicron mutant, by approximately 1,500- fold, and one of the mutations led to an improvement in activity against multiple variants to a certain extent. Together, we established a procedure of rapid selection of neutralizing antibodies with potent SARS-CoV-2 neutralization activity. Our results provide a reference for engineering neutralizing antibodies against future SARS-CoV-2 variants and even other pandemic viruses.

Domain structure of ENPP1 and CDH11.

Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63-69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Therapeutic antibody design has garnered widespread attention, highlighting its interdisciplinary importance. Advancements in technology emphasize the critical role of designing nanobodies and humanized antibodies in antibody engineering. However, current experimental methods are costly and time-consuming. Computational approaches, while progressing, faced limitations due to insufficient structural data and the absence of a standardized protocol. To tackle these challenges, our lab previously developed IsAb1.0, an in silico antibody design protocol. Yet, IsAb1.0 lacked accuracy, had a complex procedure, and required extensive antibody bioinformation. Moreover, it overlooked nanobody and humanized antibody design, hindering therapeutic antibody development. Building upon IsAb1.0, we enhanced our design protocol with artificial intelligence methods to create IsAb2.0. IsAb2.0 utilized AlphaFold-Multimer (2.3/3.0) for accurate modeling and complex construction without templates and employed the precise FlexddG method for in silico antibody optimization. Validated through optimization of a humanized nanobody J3 (HuJ3) targeting HIV-1 gp120, IsAb2.0 predicted five mutations that can improve HuJ3-gp120 binding affinity. These predictions were confirmed by commercial software and validated through binding and neutralization assays. IsAb2.0 streamlined antibody design, offering insights into future techniques to accelerate immunotherapy development.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

Fig S1: Site density cell lines with cytokine analyses of normal healthy donors and patient-derived CD22 CART. Fig S2: Development and evaluation of high-affinity CD22V1 CAR. Fig S3: PKCB2 effects of Bryostatin1 and CD22 modulation with PKC-inhibitors. Fig S4: RNA-seq analysis of Bryostatin1 effects on KOPN8 and SEM Fig S5: Exhaustion markers in Bryostatin1 and CD19 CAR with Bryostatin1 Fig S6: Bioluminescence for Bryostatin1 in vivo experiments Table S1: Enrichment analysis of RNA-seq Bryostatin1-treated cells specific to intrinsic components of the plasma membrane

PDF file, 330KB, Schematic representation and SDS-PAGE analysis of the IGF-II-specific antibodies.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

Supplementary Data from FGFR4-Targeted Chimeric Antigen Receptors Combined with Anti-Myeloid Polypharmacy Effectively Treat Orthotopic Rhabdomyosarcoma

Supplementary Table from Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

PDF file - 782K, Binding of the SS1, HN1 or SD1 VH domain to mesothelin (MSLN).