Single-chain Antibody Fragment Research Articles

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes type II

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1β. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments.

Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.

Pharmacokinetic evaluation of a single chain antibody fragment against scorpion toxins in sheep

A key aspect during the development of antivenoms is the evaluation of the efficiency and security of the therapeutic molecules. In this work, we report the pharmacokinetic analysis of a neutralizing single chain antibody fragment named LR (scFv LR) where three sheep were used as a large animal model. The animals were injected through i.v. route with 2 mg of scFv LR. Blood samples were drawn every minute within the first 15 min, the sampling continues at 20, 25, 30, 45, 60, 90, 120 min, subsequently at 1-h intervals, 3, 4, 5, 6 h, two more samples at 9 and 12 h and, two more samples at 24 and 48 h and finally at one-day intervals during 4 days. scFv LR levels were measured from blood serum and urine samples by an ELISA. The pharmacokinetics of the experimental data was analyzed using the three-exponential kinetics. The value of the fast initial component (τ1=0.409±0.258min) indicated that the scFv is distributed rapidly into the tissues. The mean residence time, MRT, was 45 ± 0.51 min and the clearance (CL), 114.3 ± 14.3 mL/min. From urine samples it was possible to detect significant amounts of scFv LR, which is evidence of renal elimination.

Abstract 3598: A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager

Abstract TCR-engineered T (TCR-T) cells and TCR-anti-CD3 bispecific T-cell engagers (TCEs) are potent TCR-based therapeutic agents with distinct advantages and limitations in tumor treatment. TCR-T cells offer durable persistence within patients but necessitate personalized manufacturing and lack the capacity to harness bystander T cells. Conversely, TCEs are readily available as "off-the-shelf" products and can recruit bystander T cells, yet they exhibit a shorter lifespan. In our study, we sought to merge the merits of both approaches by engineering T cells to secrete a TCR-anti-CD3 TCE specific for alpha fetoprotein (AFP), a tumor-associated antigen abundantly expressed in hepatocellular carcinoma (HCC). We initially identified a TCR with specificity for the AFP158-166 peptide bound to HLA-A*02:01 and enhanced its affinity to picomolar via phage display. To facilitate efficient secretion by T cells, we adapted the high-affinity TCR to a single-chain format (scTCR) and fused it with a CD3-specific single-chain antibody fragment (scAFP-TCE). Our findings demonstrated that scAFP-TCE effectively redirected bystander T cells to engage in the lysis of HCC cells. Moreover, scAFP-TCE could be secreted by T cells transduced with lentiviral particles encoding the TCE gene. These transduced T cells exhibited potent antitumor activity both independently and by enlisting bystander T cells. This innovative T cell strategy, combining the bystander-recruiting ability of fusion proteins with the durable persistence seen in T cell therapy after a single infusion, presents a promising alternative to conventional TCR-based therapeutic agents. We anticipate that this novel approach may hold substantial potential for enhancing HCC treatment and expanding the scope of TCR-based immunotherapies. Citation Format: Hanli Sun, Jiao Huang, Kai Zhan, Wanli Wu, Xianqing Tang, Min Liu, Shanshan Guo, Hongjun Zheng, Yingjie Huang, Shi Zhong. A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3598.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV)

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Biotin as a structural component in the detection of small model antigens in BLA-S-ELISA

An interesting biotin-linked-antigen-Sandwich-ELISA was developed (BLA-S-ELISA), which based on the captured Trinitrophenol-Biotin (TNP-Biotin) molecule between the immobilised monovalent antibody and enzyme-conjugated streptavidin. Monoclonal anti-Trinitrotoluene single chain fragment antibody (anti-TNT-scFv) was cloned and expressed in E. coli cells, and then used as an immobilised component in an assay. Thereafter, the previously synthesised TNP-Biotin was added as antigen followed by the addition of streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate which led finally to the formation of a three-component system (antibody/TNP-Biotin/streptavidin-HRP). The assay was performed with a range of different dilutions of TNP-Biotin to establish its minimal detectable concentration. The detection limit of TNP-Biotin was 4 ngmL−1 (i.e. 200 pg or 0.42 pmol antigen calculated on the basis of 50 μL sample or 8.4 nM expressed in concentration units). According to our best knowledge, this is the very first time for any model antigen to be detected with such a form of biotin-streptavidin sandwich-assay.

THE OUTCOME OF INTRAVITREAL BROLUCIZUMAB FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION TREATMENT

Introduction : Age-related macular degeneration (AMD) is a disease that results in irreversible severe loss of central vision in people over 50 years old. Hence, anti-VEGF therapy has become a standard treatment for neovascular AMD. Brolucizumab is a single-chain antibody fragment that inhibits VEGF-A binding to VEGF receptors. It has a higher molar concentration that allows a greater reduction in the central subfield thickness (CST)
 Case Illustration : A 59-year-old male with a chief complaint of gradually blurred vision in the left eye for 2 years. BCVA of the left eye was 0.3, OCT showed a hyperreflective band in the sub-neurosensory space with subretinal fluid in the left eye (Figure 1), and OCT-A revealed choroidal neovascularization (Figure 2). The patient was diagnosed with AMD AREDS IV in the left eye. He had undergone three monthly injections of brolucizumab. BCVA of the left eye after injection improved to 1.0 and OCT imaging showed no subretinal fluid, no intraretinal fluid, and no pigment epithelial detachment.
 Discussion : Brolucizumab has a greater reduction in CST because of its high molecular concentration. Results of HAWK and HARRIER concluded that brolucizumab was non-inferior towards aflibercept in BCVA change at week 48. According to the HAWK trial, a greater reduction of CST was seen in week 16 among eyes treated with brolucizumab. Brolucizumab was also well tolerated and adverse event rates were similar to aflibercept.
 Conclusion : Brolucizumab could be an alternative treatment option considering its efficacy in improving visual acuity and reducing CST.

SON-1210 - a novel bifunctional IL-12 / IL-15 fusion protein that improves cytokine half-life, targets tumors, and enhances therapeutic efficacy.

The potential synergy between interleukin-12 (IL-12) and IL-15 holds promise for more effective solid tumor immunotherapy. Nevertheless, previous clinical trials involving therapeutic cytokines have encountered obstacles such as short pharmacokinetics, limited tumor microenvironment (TME) targeting, and substantial systemic toxicity. To address these challenges, we fused single-chain human IL-12 and native human IL-15 in cis onto a fully human albumin binding (FHAB) domain single-chain antibody fragment (scFv). This novel fusion protein, IL12-FHAB-IL15 (SON-1210), is anticipated to amplify the therapeutic impact of interleukins and combination immunotherapies in human TME. The molecule was studied in vitro and in animal models to assess its pharmacokinetics, potency, functional characteristics, safety, immune response, and efficacy. SON-1210 demonstrated robust binding affinity to albumin and exhibited the anticipated in vitro activity and tumor model efficacy that might be expected based on decades of research on native IL-12 and IL-15. Notably, in the B16F10 melanoma model (a non-immunogenic, relatively "cold" tumor), the murine counterpart of the construct, which had mouse (m) and human (h) cytokine sequences for the respective payloads (mIL12-FHAB-hIL15), outperformed equimolar doses of the co-administered native cytokines in a dose-dependent manner. A single dose caused a marked reduction in tumor growth that was concomitant with increased IFNγ levels; increased Th1, CTL, and activated NK cells; a shift in macrophages from the M2 to M1 phenotype; and a reduction in Treg cells. In addition, a repeat-dose non-human primate (NHP) toxicology study displayed excellent tolerability up to 62.5 µg/kg of SON-1210 administered three times, which was accompanied by the anticipated increases in IFNγ levels. Toxicokinetic analyses showed sustained serum levels of SON-1210, using a sandwich ELISA with anti-IL-15 for capture and biotinylated anti-IL-12 for detection, along with sustained IFNγ levels, indicating prolonged kinetics and biological activity. Collectively, these findings support the suitability of SON-1210 for patient trials in terms of activity, efficacy, and safety, offering a promising opportunity for solid tumor immunotherapy. Linking cytokine payloads to a fully human albumin binding domain provides an indirect opportunity to target the TME using potent cytokines in cis that can redirect the immune response and control tumor growth.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4–15 (scFv 4–15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4–15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

Design of a Superior Factor VIIIa Mimetic By Coupling a Membrane Binding Domain to a Factor IXa Binding Antibody Fragment

Emicizumab is a bispecific antibody that augments Factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Clinical use of this FVIIIa mimetic significantly reduces bleeding episodes in Hemophilia A (HA) patients. Membrane dependent assembly of FVIIIa-FIXa-FX is an absolute requirement for the intrinsic tenase activity. In contrast, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation even in the absence of membranes. This major distinction likely allows FVIIIa to function at sub-nanomolar concentrations to greatly accelerate the activation of FX while only much lower enhancements of rate are achieved at several 100-fold higher concentrations of emicizumab. We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity obtained with the bispecific antibody. We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (V H) with the variable light chain (V L) to produce V H9V L. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, V H9V L, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, V H9V L, was used as a template to introduce membrane binding properties. The Factor V C2-domain, which binds membrane, was fused at the C-terminus of V H9V L via a repeating Gly 4Ser flexible linker of ~ 5.7 nm to allow the V H and V L domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct V H9V LC2 showed a substantial amplification in cofactor mimetic activity as compared to V H9V L. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by V H9V LC2 as compared to V H9V L. Remarkably, despite its inability to bind FX, cofactor mimetic activity of V H9V LC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by V H9V LC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). V H9V LC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM). The role of C2 domain-mediated increase in cofactor activity of V H9V LC2 was further assessed by using an antibody fragment (E 9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E 9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of V H9V LC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E 9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of V H9V LC2. To evaluate whether V H9V LC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of V H9V LC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that V H9V LC2 can bypass FVIII inhibitor activity. The engineered scFv with a factor V C2 domain fusion, V H9V LC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.

SURG-09. TARGETING A SHARED GLIOBLASTOMA CELL STATE THROUGH ADAPTIVE NATURAL KILLER CELLS AUGMENTED WITH TRISPECIFIC KILLER CELL ENGAGER (TRIKE) IMMUNE-MODULATOR AGAINST CD276

Abstract INTRODUCTION T and NK (Natural Killer) cells have emerged as promising platforms for immunotherapy. While T cells require antigen priming and complex MHC-dependent interactions, NK cells exhibit limited persistence and limited antigen specificity. Trispecific Killer cell Engagers (TriKEs) are constructs developed to address these limitations, combining interleukin 15 (IL15, to enhance persistence), a single-chain antibody fragment against CD16 (to immune-activate NK), and a second fragment targeting a tumor surface marker. Objective: In this study, we identified CD276 (B7H3) as the surface marker optimal for an anti-glioblastoma TriKE and tested its effect on the anti-glioblastoma activity of NK cells. Method: Mass-spectrometry, RNA-Seq, and single cell RNA-Seq profiling of glioblastoma lines. Laboratory characterization and murine modeling Result: Orthogonal analysis of mass-spec identified cell surface molecules enriched in glioblastoma lines and RNA-seq of lines with defined sensitivity to NK cells identified surface proteins highly expressed in NK-resistant lines. Screening of these candidates for genes that showed poor survival association using the TCGA identified CD276. qPCR analysis confirmed higher CD276 expression in the NK-resistant lines, though expression is also detected in NK-sensitive glioblastoma lines. Western blotting analysis and TCGA analysis showed higher level CD276 expression in all glioblastoma subtypes (proneural, neural, mesenchymal, and classical) relative to normal brain. Single cell RNA-Seq of clinical specimens confirmed high expression of CD276 in glioblastoma cells in OPC-like, NPC-like, AC-like, and MES-like cell states. Co-incubation of a IL15, CD16, and CD276 targeting TriKE with either adaptive or iPS derived NK cells enhanced anti-glioblastoma cytotoxicity against cells otherwise refractory to NK treatment in vitro. Synergy between these reagents were also observed in vivo upon orthotopic injection. CONCLUSION Our results suggest that a therapeutic strategy combining intra-tumoral injections of NK cells and a B7H3 specific TriKE holds promise and warrants clinical trial consideration.

CTIM-34. JACOB: JOINT ADMINISTRATION OF CDNA FOR TP53, CHECKPOINT INHIBITION, AND BOOST/HYPO FRACTIONATED RADIATION. A PHASE 0/1 STUDY IN CHILDREN WITH RECURRENT, PROGRESSIVE, OR REFRACTORY CNS MALIGNANCIES

Abstract BACKGROUND Pediatric patients with recurrent, progressive, or refractory brain tumors have dismal prognoses and few therapeutic options. Due to the complex biological mechanisms of resistance in these tumors, targeted agents and immunotherapeutic approaches in isolation have yielded disappointing results. The combination of SGT-53, anti-PD-1, and hypofractionated radiation has shown encouraging results in preclinical studies. SGT-53 is a novel nanoparticle carrying cDNA encoding human wild-type human TP53; the nanoparticle uses a single-chain antibody fragment recognizing the human transferrin receptor, leading to excellent blood-brain barrier penetrance and anti-tumor targeting. TP53 therapy work synergistically with immune checkpoint inhibitors like anti-PD-1. Hypofractionated radiation provides additional synergy, increasing vascular access, cytokine production and activating tumor-specific T-cells. METHODS JACOB is a prospective, phase 0/1 clinical trial to evaluate the safety and efficacy of SGT-53 in combination with hypofractionated radiation and anti-PD-1 in children with recurrent, progressive, or refractory CNS malignancies. Inclusion criteria: patients age ≥ 3 and ≤ 21 years, measurable disease, performance status >50 and adequate organ function. The 3 + 3 de-escalation design will be used to determine the maximum tolerated dose (MTD) of SGT-53 in combination with ICI and radiation. Dose limiting toxicities will be evaluated. SGT-53 will be administered two times per week. Hypofractionated radiation will be given in week 2, and anti-PD-1 (once in 2 weeks) will begin on the second week of treatment. A maintenance phase of SGT-53 and anti-PD-1 will be continued for up to 12 months. Primary endpoints evaluate toxicities related to SGT-53 and plasma pharmacokinetics. Secondary and exploratory endpoints include best radiological response, and immunobiological markers. Twelve patients will be treated in the phase I/dose de-escalation cohort. After the MTD has been identified, a target validation/ phase 0 cohort will include up to 4 patients receiving 2 doses of SGT-53 prior to undergoing surgical resection to determine tissue-specific response.

EGFR-targeted humanized single chain antibody fragment functionalized silica nanoparticles for precision therapy of cancer

The combination of highly specific targeting ability and potent killing effect has made antibody-drug conjugates (ADCs) a popular area of focus in the development of anti-cancer drugs. However, the large molecular weight of IgG antibodies (∼ 150 kDa) often faces challenges in penetrating capillaries and stroma in tumor tissue. Moreover, when the drug-antibody ratio (DAR) is too low (DAR < 2) or too high (DAR > 6) it decreases the effectiveness of the ADC and further increases the potential for aggregation, overall clearance of the early system payload, and release rate. In this study, an EGFR-based single-chain antibody fragment (husA)-human serum albumin (HSA)-coupled FITC-labeled mesoporous silica nanoparticle (FMSN-DOX-H-husA) was developed. Chinese hamster ovarian cells express the husA, which is a single chain antibody fragment of the EGFR that has been humanized. The small molecular weight of the single chain antibody allows for shorter penetration into solid tumors and the absence of adverse effects of the Fc fragment. The modification of HSA improves the safety of the antibody nanoparticle couples by both improving the biocompatibility of the nanoparticles, prolonging the circulation time of the nanoparticles, and avoiding early release of the payload. Also, the humanization substantially reduces the immunogenicity. More importantly, the ratio of drug antibodies on nanoparticles was experimentally and computationally derived to be 11.8, providing a more accurate guide for clinical trials. The results of both in vivo and in vitro experiments indicated promising antitumor activity and safety of FMSN-DOX-H-husA. Thus, this antibody-drug conjugate provided a hopeful option for cancer treatment.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody–antigen interactions. The method, which we named ‘deep screening’, involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument’s flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

Single chain antibody fragment display systems: a review

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Single-chain fragment antibody disrupting the EphA4 function as a therapeutic drug for gastric cancer

Studies have shown that the high expression of EphA4 in gastric cancer tissues may correlate with unfavorable clinical pathological characteristics. Therefore, EphA4 may be an effective target for treating gastric cancer in addition to HER-2/neu. In this study, generated scFv S3 can bind endogenous EphA4 of gastric cancer cells and has significant membrane staining. Additionally, scFv S3 binding to EphA4 inhibits the growth and migration of cancer cells and the growth induction that ephrinA1 generates in gastric cancer cells. We found that EphA4 molecules may degrade through antibody treatment of cells, and the increase in LAMP1 and LAMP2 indicates that lysosome is involved in the degradation. The scFv S3 administration leads to the signals pAKT, pERK, and pSTAT3 decrease in cancer cells. The xenograft model of HER-2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than trastuzumab scFv. The scFv S3 administration in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved C3 molecule expression. Furthermore, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. The antibody may provide effective treatment for patients with gastric cancer and abnormal activation or overexpression of EphA4 signaling.

Intravitreal RTH258 (brolucizumab) demonstrates no effect on pregnancy, parturition, embryofetal or postnatal development in cynomolgus monkeys

RTH258 (brolucizumab) is a humanized single chain antibody fragment, the smallest functional unit of an antibody designed to target vascular endothelial growth factor in angiogenic retinal disease. To further understand the safe use of RTH258, this study assessed the potential impact of intravitreal RTH258 on pre- and postnatal development in the offspring of cynomolgus monkeys following administration to the mother. Three groups of 16 pregnant females were included: a low dose group (RTH258 3 mg/50 µl [60 mg/ml]), a high dose group (RTH258 6 mg/50 µl [120 mg/ml]), and a control group. Maternal animals were administered a single injection of 50 µl in the right eye once every four weeks. Animals were observed daily and detailed observations were collected before and after the first dose, and then weekly thereafter. Following parturition, observations of infants included external, morphological, and ophthalmic examinations; neurobehavioral test battery; grip strength; and skeletal development. Blood samples for hematology, coagulation, and clinical chemistry were collected from non-fasted maternal and infant animals. No RTH258-related deaths occurred in maternal dams or infants. No RTH258-related clinical observations were noted in maternal animals or in surviving infants - there were no changes in gestation length; pregnancy loss; deaths; body weight/weight change; infant grip strength; infant external, morphological, or skeletal evaluations; ophthalmoscopy or neurobehavioral observations; or clinical pathology parameters. RTH258 had no impact on pregnancy or parturition; embryo-fetal development; or survival, growth, or postnatal development of offspring when administered via repeated intravitreal administration.

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE.

The global prevalence of β-lactam allergy poses a major challenge in administering first-line antibiotics, such as penicillins, to a significant portion of the population. The lack of β-lactam IgE antibody pools with defined selectivity hampers the standardization and validation of in vitro assays for β-lactam allergy testing. To address this limitation, this study introduces a synthetic IgE specific to β-lactam antibiotics as a valuable tool for drug allergy research and diagnostic tests. Using phage display technology, we constructed a library of human single-chain antibody fragments (scFv) to target the primary determinant of amoxicillin, a widely used β-lactam antibiotic. Subsequently, we produced a complete human synthetic IgE molecule using the highly efficient baculovirus expression vector system. This synthetic IgE molecule served as a standard in an in vitro chemiluminescence immunoassay for β-lactam antibiotic allergy testing. Our results demonstrated a detection limit of 0.05 IU/mL (0.63 pM), excellent specificity (100%), and a four-fold higher clinical sensitivity (73%) compared to the in vitro reference assay when testing a cohort of 150 serum samples. These findings have significant implications for reliable interlaboratory comparison studies, accurate labeling of allergic patients, and combating the global public health threat of antimicrobial resistance. Furthermore, by serving as a valuable trueness control material, the synthetic IgE facilitates the standardization of diagnostic tests for β-lactam allergy and demonstrates the potential of utilizing this synthetic strategy as a promising approach for generating reference materials in drug allergy research and diagnostics.

Enhanced antitumor and anti-metastasis by VEGFR2-targeted doxorubicin immunoliposome synergy with NK cell activation.

Liposomal doxorubicin exhibits stronger drug accumulation at the tumor site due to the Enhanced Permeability and Retention (EPR) effect. However, the prognosis for the patient is poor due to this drug's lack of targeting and tumor metastasis during treatment. Vascular epidermal growth factor receptor (VEGFR2) plays an important role in angiogenesis and cancer metastasis. To enhance antitumor efficacy of PEGylated liposomal doxorubicin, we constructed a VEGFR2-targeted and doxorubicin-loaded immunoliposome (Lipo-DOX-C00) by conjugating a VEGFR2-specific, single chain antibody fragment to DSPE-PEG2000-MAL, and then we inserted the antibody-conjugated polymer into liposomal doxorubicin (Lipo-DOX). The immunoliposome was formed uniformly with high affinity for VEGFR2. In vitro, Lipo-DOX-C00 enhanced doxorubicin internalization into LLC and 4T1 cells compared with non-conjugated, liposomal doxorubicin. In vivo, Lipo-DOX-C00 delivered DOX to tumor tissues effectively, which exhibited an improved antitumor and anti-metastasis efficacy in both LLC subcutaneous tumor models and 4T1 tumor models. In addition, the combined therapy of a VEGFR2-MICA bispecific antibody (JZC01) and Lipo-DOX-C00 achieved enhanced inhibition of cancer growth and metastasis due to activation of the immune system. Our study provides a promising approach to clinical application of liposomal doxorubicin.