Biology subjects

Dimitrov, D. S.

Publications and source records attributed to Dimitrov, D. S..

9 recordsLinked to original sources

bioRxiv2026

Potent and Broad HIV-1 Neutralization by a Bispecific CD4-CD4i Fusion Protein based on Single-Domain CD4-D1 and X5 CD4i antibody

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 g/mL and 3.4 g/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.

molecular biology↗

bioRxiv2025

KDM5A enhances MHC-I antigen presentation and potentiates antitumor immunity unleashed by immune checkpoint blockade

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-{gamma}/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.

immunology↗

bioRxiv2022

Discovery and pre-clinical evaluation of antibodies to the NKG2A inhibitory receptor

NK and CD8+ T cells are important cells for cytolysis of cancer cells. The tumor microenvironment can upregulate surface expression on these cells of NKG2A, an inhibitory receptor that can dampen immune responses to cancer leading to immune evasion. To block NKG2A-mediated inhibition, we discovered and characterized two fully human antibodies using phage and yeast display that bind to NKG2A. These antibodies are highly specific for human CD94/NKG2A heterodimer complex, displaying no binding to the activating NKG2C receptor. A mutagenesis study revealed that the serine residue at 170 position (S170) of NKG2A is critical for the selectivity of anti-NKG2A antibodies. In vitro cytotoxic assays showed that NKG2A antibody inhibitors activated primary NK cells and promoted ADCC function of specific antibodies that bind to antigens expressed on cancer cells. Summary headingFully human antibodies to the NKG2A inhibitory receptor

immunology↗

bioRxiv2022

Discovery of a novel mesothelin specific human antibody VH domain

Antibody based therapeutics targeting mesothelin (MSLN) have shown limited anticancer activity in clinical trials. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel VH domain 3C9 from a large size human immunoglobulin heavy chain variable (VH) domain library. 3C9 exhibited high affinity [KD (dissociation constant) < 3 nM] and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human Fc became visible at tumor sites as early as 8 hours post infusion and persisted at the tumor site for more than 10 days. Both CAR-T cells and antibody domain drug conjugations (DDCs) generated with 3C9 were highly effective at killing MSLN positive cells in vitro without off-target effects. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residues patches on the MSLN surface. 3C9 fused to human Fc domain drug conjugate was efficacious to inhibit tumor growth in a mouse xenograft model. This newly discovered VH antibody domain holds promise as a therapeutic candidate for MSLN-expressing cancers.

cancer biology↗

bioRxiv2022

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

immunology↗

bioRxiv2021

Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding

The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

biochemistry↗

bioRxiv2021

Neutralization of European, South African, and United States SARS-CoV-2 mutants by a human antibody and antibody domains

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission with several emerging variants remain uncontrolled in many countries, indicating the pandemic remains severe. Recent studies showed reduction of neutralization against these emerging SARS-CoV-2 variants by vaccine-elicited antibodies. Among those emerging SARS-CoV-2 variants, a panel of amino acid mutations was characterized including those in the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. In the present study, we evaluated our previously identified antibody and antibody domains for binding to these RBD variants with the emerging mutations, and neutralization of pseudo typed viruses carrying spike proteins with such mutations. Our results showed that one previously identified antibody domain, ab6, can bind 32 out of 35 RBD mutants tested in an ELISA assay. All three antibodies and antibody domains can neutralize pseudo typed B.1.1.7 (UK variant), but only the antibody domain ab6 can neutralize the pseudo typed virus with the triple mutation (K417N, E484K, N501Y). This domain and its improvements have potential for therapy of infections caused by SARS-CoV-2 mutants.

immunology↗

bioRxiv2021

Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody

The recently reported "UK variant" of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-[A] resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments. Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

biophysics↗

bioRxiv2020

Potent neutralization of SARS-CoV-2 in vitro and in an animal model by a human monoclonal antibody

Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized replication competent SARS-CoV-2 with exceptional potency as measured by two different assays. There was no enhancement of pseudovirus infection in cells expressing Fc{gamma} receptors at any concentration. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 potently neutralized mouse ACE2 adapted SARS-CoV-2 in wild type BALB/c mice and native virus in hACE2 expressing transgenic mice. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

microbiology↗