Search Results (26)

Background/Objectives: Identification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. Methods: Through single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced a BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were measured with ELISA and a pseudovirus-based neutralization assay, respectively. Their epitopes were mapped by competition ELISA and site-directed mutation. Results: Among a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13 and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20 and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. Conclusions: These four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection. Full article

Herpes simplex viruses (HSV-1 and HSV-2), which can be transmitted both orally and sexually, cause lifelong morbidity and in some cases, meningitis and encephalitis. While both the passive transfer of neutralizing antibodies and placental transfer of anti-HSV monoclonal antibodies (Mabs) have shown therapeutic promise in animal models, clinical trials have yet to identify approved immunotherapeutics for herpes infection. Here, we present strategies for the generation of recombinant bispecific antibodies (BsAbs) that target different domains of glycoprotein D (gD), crucial for HSV entry, that have the potential to outperform the effect of individual Mabs to curb herpes infection. Specifically, we selected three pairs of Mabs from our extensive panel for BsAb design and production based on their binding site and ability to block virus entry. Actual binding of BsAbs to gD and epitope availability on gD after BsAb binding were characterized using surface plasmon resonance (SPR) and inhibition by IgG Fab fragments generated from selected Mabs. While one BsAb exhibited an additive effect similar to that observed using a combination of the Mabs utilized for its generation, two showed antagonistic effects, suggesting that the simultaneous engagement of two epitopes or selective binding to one affected their activity against HSV. One BsAb (DL11/1D3) targeting the binding site for both nectin-1 and HVEM receptors demonstrated synergistic inhibitory activity against HSV, outperforming the effect of the individual antibodies. Recombinant DL11/1D3 antibody variants, in which the size of one or both paratopes was decreased to single chains (scFv-Fc), highlighted differences in potency depending on antibody size and format. We propose that BsAbs to individual glycoproteins offer a potential avenue for herpes therapeutics, but their design, mechanism of action, antibody format, and epitope engagement require careful consideration of structure for optimal efficacy. Full article

Background/Objective: Current treatments for eosinophilic and mast cell disorders are often ineffective. One promising target to improve outcomes is sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8). As limitations, there are few Siglec-8 monoclonal antibodies (mAbs) available to date, and Siglec-8-directed treatments have so far primarily focused on unconjugated mAbs, which may be inadequate, especially against mast cells. Methods: Here, we used transgenic mice to raise a diverse panel of fully human mAbs that either recognize the V-set domain, membrane-distal C2-set domain, or membrane-proximal C2-set domain of full-length Siglec-8 as a basis for novel therapeutics. Results: All mAbs were efficiently internalized into Siglec-8-expressing cells, suggesting their potential to deliver cytotoxic payloads. Tool T cell-engaging bispecific antibodies (BiAbs) and chimeric antigen receptor (CAR)-modified natural killer (NK) cells using single-chain variable fragments from Siglec-8 mAbs showed highly potent cytolytic activity against Siglec-8-positive cells even in cases of very low target antigen abundance, whereas they elicited no cytolytic activity against Siglec-8-negative target cells. Siglec-8^V-set-directed T cell-engaging BiAbs and Siglec-8^V-set-directed CAR-modified NK cells induced substantially greater cytotoxicity against cells expressing an artificial smaller Siglec-8 variant containing only the V-set domain than cells expressing full-length Siglec-8, consistent with the notion that targeting membrane-proximal epitopes enhances effector functions of Siglec-8 antibody-based therapeutics. Indeed, unconjugated Siglec-8^C2-set mAbs, Siglec-8^C2-set-directed T cell-engaging BiAbs, and Siglec-8^C2-set-directed CAR-modified NK cells showed high antigen-specific cytolytic activity against Siglec-8-positive human cell lines and primary patient eosinophils. Conclusions: Together, these data demonstrate Siglec-8-directed immunotherapies can be highly potent, supporting their further development for eosinophilic and mast cell disorders. Full article

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC₅₀ of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer. Full article

Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4, p < 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p < 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p < 0.01) and GBM12 (p < 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p < 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas. Full article

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated K_D of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated K_D of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (K_D of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo. Full article

The constant region of the immunoglobulin (IG) or antibody heavy gamma chain is frequently engineered to modify the effector properties of the therapeutic monoclonal antibodies. These variants are classified in regards to their effects on effector functions, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) enhancement or reduction, B cell inhibition by the coengagement of antigen and FcγR on the same cell, on half-life increase, and/or on structure such as prevention of IgG4 half-IG exchange, hexamerisation, knobs-into-holes and the heteropairing H-H of bispecific antibodies, absence of disulfide bridge inter H-L, absence of glycosylation site, and site-specific drug attachment engineered cysteine. The IMGT engineered variant identifier is comprised of the species and gene name (and eventually allele), the letter ‘v’ followed by a number (assigned chronologically), and for each concerned domain (e.g, CH1, h, CH2 and CH3), the novel AA (single letter abbreviation) and IMGT position according to the IMGT unique numbering for the C-domain and between parentheses, the Eu numbering. IMGT engineered variants are described with detailed amino acid changes, visualized in motifs based on the IMGT numbering bridging genes, sequences, and structures for higher order description. Full article

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency. Full article

Historically, the standard of care for hemophilia A has been intravenous administration of exogenous factor VIII (FVIII), either as prophylaxis or episodically. The development of emicizumab, a humanized bispecific monoclonal antibody mimicking activated FVIII, was a subsequent advance in treatment. However, both exogenous FVIII and emicizumab require repeated and lifelong administration, negatively impacting patient quality of life. A recent breakthrough has been the development of gene therapy. This allows a single intravenous treatment that could result in long-term expression of FVIII, maintenance of steady-state plasma concentrations, and minimization (or possibly elimination) of bleeding episodes for the recipient’s lifetime. Several gene therapies have been assessed in clinical trials, with positive outcomes. Valoctocogene roxaparvovec (an adeno-associated viral 5-based therapy encoding human B domain-deleted FVIII) is expected to be the first approved gene therapy in European countries, including Italy, in 2022. Some novel challenges exist including refining patient selection criteria, managing patient expectations, further elucidation of the durability and variability of transgene expression and long-term safety, and the development of standardized ‘hub and spoke’ centers to optimize and monitor this innovative treatment. Gene therapy represents a paradigm shift, and may become a new reference standard for treating patients with hemophilia A. Full article

B-cell lymphoma is a group of malignant proliferative diseases originating from lymphoid tissue with different clinical manifestations and biological characteristics. It can occur in any part of the body, accounting for more than 80% of all lymphomas. The present study aimed to construct bispecific single-domain antibodies against CD20 and CD3 and to evaluate their function in killing tumor cells in vitro. A Bactrian camel was immunized with a human CD20 extracellular peptide, and the VHH gene was cloned and ligated into a phagemid vector to construct the phage antibody display library. A phage antibody library with a size of 1.2 × 10⁸ was successfully constructed, and the VHH gene insertion rate was 91.7%. Ninety-two individual clones were randomly picked and screened by phage ELISA. Six strains with the high binding ability to human CD20 were named 11, 30, 71, 72, 83, and 92, and induced expression and purification were performed to obtain soluble CD20 single-domain antibodies. The obtained single-domain antibodies could specifically bind to human CD20 polypeptide and cell surface-expressed CD20 molecules in ELISA, Western blot, and cell immunofluorescence assays. The anti-CD20/CD3 bispecific nanobody (BsNb) was successfully constructed by fusing the anti-CD20 VHH gene with the anti-CD3 VHH and the bispecific single-domain antibody was expressed, purified, and validated. Anti-CD20/CD3 BsNb can specifically bind CD20 molecules on the surface of human lymphoma Raji cells and CD3 molecules on the surface of T cells in flow cytometry analysis and effectively mediate peripheral blood mononuclear cells (PBMCs) target Raji cells with a killing efficiency of up to 30.4%, as measured by the lactate dehydrogenase (LDH) method. The release of hIFN-γ from PBMCs during incubation with anti-CD20/CD3 BsNb was significantly higher than that of the control group (p < 0.01). The anti-CD20/CD3 BsNb could maintain 80% binding activity after incubation with human serum at 37 °C for 48 h. These results indicated the strong antitumor effect of the constructed anti-CD20/CD3 BsNb and laid the foundation for the further development of antitumor agents and the clinical application of anti-CD20/CD3 BsNb. Full article

STAT3 and KRAS regulate cell proliferation, survival, apoptosis, cell migration, and angiogenesis. Aberrant expression of STAT3 and mutant active forms of KRAS have been well-established in the induction and maintenance of multiple cancers. STAT3 and KRAS mutant proteins have been considered anti-cancer targets; however, they are also considered to be clinically “undruggable” intracellular molecules, except for KRAS(G12C). Here we report a first-in-class molecule, a novel, single domain camelid VHH antibody (15 kDa), SBT-100, that binds to both STAT3 and KRAS and can penetrate the tumor cell membrane, and significantly inhibit cancer cell growth. Additionally, SBT-100 inhibits KRAS GTPase activity and downstream phosphorylation of ERK in vitro. In addition, SBT-100 inhibits the growth of multiple human cancers in vitro and in vivo. These results demonstrate the feasibility of targeting hard-to-reach aberrant intracellular transcription factors and signaling proteins simultaneously with one VHH to improve cancer therapies. Full article

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents. Full article

The ability to kill tumor cells while maintaining an acceptable safety profile makes Natural Killer (NK) cells promising assets for cancer therapy. Strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment can be expected to increase the efficacy of NK cell-based therapies. In this study, we show binding of a novel bispecific single domain antibody (VHH) to both CD16 (FcRγIII) on NK cells and the epidermal growth factor receptor (EGFR) on tumor cells of epithelial origin. The bispecific VHH triggered CD16- and EGFR-dependent activation of NK cells and subsequent lysis of tumor cells, regardless of the KRAS mutational status of the tumor. Enhancement of NK cell activation by the bispecific VHH was also observed when NK cells of colorectal cancer (CRC) patients were co-cultured with EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the bispecific VHH and autologous peripheral blood mononuclear cells or allogeneic CD16 expressing NK cells. The anticancer activity of CD16-EGFR bispecific VHHs reported here merits further exploration to assess its potential therapeutic activity either alone or in combination with adoptive NK cell-based therapeutic approaches. Full article

The increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells. Full article

The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3⁺ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC₅₀ in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies. Full article