Epitope Binning Research Articles

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells.

Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9

Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.

A broadly protective antibody targeting glycoprotein Gn inhibits severe fever with thrombocytopenia syndrome virus infection

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes severe viral hemorrhagic fever and thrombocytopenia syndrome with a fatality rate of up to 30%. No licensed vaccines or therapeutics are currently available for humans. Here, we develop seven monoclonal antibodies (mAbs) against SFTSV surface glycoprotein Gn. Mechanistic studies show that three neutralizing mAbs (S2A5, S1G3, and S1H7) block multiple steps during SFTSV infection, including viral attachment and membrane fusion, whereas another neutralizing mAb (B1G11) primarily inhibits the viral attachment step. Epitope binning and X-ray crystallographic analyses reveal four distinct antigenic sites on Gn, three of which have not previously been reported, corresponding to domain I, domain II, and spanning domain I and domain II. One of the most potent neutralizing mAbs, S2A5, binds to a conserved epitope on Gn domain I and broadly neutralizes infection of six SFTSV strains corresponding to genotypes A to F. A single dose treatment of S2A5 affords both pre- and post-exposure protection of mice against lethal SFTSV challenge without apparent weight loss. Our results support the importance of glycoprotein Gn for eliciting a robust humoral response and pave a path for developing prophylactic and therapeutic antibodies against SFTSV infection.

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

Abstract PO5-04-02: Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a key role in proliferation stimulator for several human cancers. In particular, HER2 is overexpressed in 15-30% of breast cancers with or without gene amplification with prognostic and predictive implications. HER2 overexpression is associated with shorter disease-free and overall survivals, resistance to hormonal agents and increased risk of brain metastasis. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular domain and blocks its signaling. More recently, two antibody drug conjugates using trastuzumab have been approved: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu to deliver the topoisomerase inhibitor deruxtecan. We are reporting here the development of fully human internalizing anti-HER2 antibodies with distinct epitopes on the HER2protein which compete or not with trastuzumab for binding to HER2. These monoclonal antibodies have been developed by immunizing fully human (TC-Mab mouse) mice with recombinant HER2 protein. After production of hybridoma secreting fully human immunoglobulins, the screening process included competition with trastuzumab for binding to Her2 by enzyme linked immunoassay and Octet epitope binning and affinity determination as well as internalization assay. Several internalizing antibodies able to compete or not with trastuzumab and with high affinity (Kd ranging from 10−9 M to 10−12 M) were selected. Their ability to deliver a cytotoxic payload in HER2 overexpressing breast cancer cells such as SKBR3, BT474 and AU565 was next investigated. Two groups of antibodies: one competing with trastuzumab and the other one non-competing were further characterized. Data related to these antibodies’ biochemical characteristics as well as their ability to inhibit proliferation in vitro and in vivo in mouse xenografts studies will be presented here. In conclusion, the use of fully human mice to develop monoclonal antibodies provides a powerful and attractive approach to develop fully human monoclonal antibodies against cancer targets by-passing the need for humanization and affinity maturation of antibodies. This work is supported by 5R44CA224718 SBIR grant from the National Cancer Institute. Citation Format: Ginette Serrero, Binbin Yue, Jianping Dong, Jun Hayashi. Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-02.

Abstract 7532: NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells

Abstract Introduction: Immune checkpoint blockers (ICBs) have revolutionized treatment, enhancing survival and benefiting many patients. However, many patients fail to respond to the treatment, necessitating research for new targets and improved approaches. The Killer cell Lectin-like Receptor G1 (KLRG1), expressed on a subset of NK and T-cells harbors an immune receptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain, indicating its inhibitory function. On CD8+ T-cells, KLRG1 is expressed on highly differentiated antigen-specific effector memory T-cells (TEMs) and CD45RA+ effector memory T cells (TEMRA). These cells are known to exhibit strong cytotoxicity potential and display developmental plasticity following KLRG1 blockade. And 16-48% of CD8+ T-cells that infiltrate the tumor microenvironment (TME) are KLRG1 positive. We hypothesize that the blockade of KLRG1 should enhance the effector function of KLRG1+ CD8 T-cells in the TME and provide therapeutic benefit as a standalone or in combination with existing ICBs. Methods: Monoclonal antibodies (mAbs) targeting the human KLRG1 extracellular domain (ECD) were generated through mouse immunization and hybridoma technology. Purified mAbs from positive clones were screened using CHO-K1 and Jurkat cells overexpressing human or cynomolgus KLRG1 and human primary CD8 T-cells (n=3) to determine EC50 values. Epitope binning was performed, and high-affinity clones from each bin with cyno cross-reactivity were humanized to IgG1-Fc LALA variant. The Abs were evaluated for ligand blocking and enhancement of CD8 T-cell effector function through multiple functional assays. The assay measured enhancement of IFNγ or TNFα by KLRG1+ human CD8+ T-cells co-cultured with CHO-K1 expressing OKT3 and E-cadherin as artificial antigen-presenting cells (aAPCs) or PBMCs stimulated in with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. A human IgG1-LALA antibody (Ab) served as a negative control. PD-1 and KLRG1 synergy was evaluated in hPBMC assays. Results: NB005.1 demonstrated single digit nanomolar (nM) binding potency on human primary CD8 T-cells and Jurkat cells expressing human KLRG1 as identified by its EC50 value. Blocking of KLRG1 with NB005.1 on purified human CD8 T-cells in co-culture assay with aAPC expressing E-cad and OKT3 led to CD8 T-cell activation and increase in IFNγ production confirming inhibitory function of KLRG1. Similar result was obtained when the Ab was tested on hPBMCs stimulated with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. Conclusion: NB005.1, a fully humanized mAb, binds to human KLRG1 in the single digit nM range and effectively blocks the interaction with its ligand. This blockade enhances T-cell effector function as measured by IFNγ production, supporting the advancement of NB005.1 for preclinical testing, either as a standalone therapeutic or in combination with existing ICBs. Citation Format: Bishnu P. Nayak, Meiguang Xiong, Yajuan Xue, Binbin Wang, Shahrzad Lighvani, Shilpi Sharma, Dong Wang, Haojai Wang, Baiyang Wang, Yu Zhang, Xin Dong. NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells [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 7532.

Abstract 5793: Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling

Abstract Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the β-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. DKK1 has been shown to be a potent inhibitor of Wnt signaling via competing Wnt binding to LRP5/6. DKK1 is tumorigenic in multiple cancer types and also immunosuppressive via MDSCs and NK cells. Emerging evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. With Twist’s precision DNA writing technologies, we have created phage display libraries with diversity greater than 1 × 1010, and utilized machine learning model for optimal discovery. In this study, we discovered anti-DKK1 antibodies bind to different cysteine-rich domain (CRD) of hDKK1 by epitope binning and mapping, and block the binding of DKK1 to the receptor. Binding of the antibodies to different CRDs of hDKK1 lead to different activation effect. The in vitro functional assays showed that the interaction of Wnt to its receptor was restored in the presence of anti-DKK1 antibodies binding to DKK1 CRD2, resulting in TCF/LEF signaling upregulation. Moreover, anti-DKK1 antibodies binding to DKK1 CRD1 induced immune cell activation and led to tumor cell cytotoxicity. In vivo study indicated anti-DKK1 antibody lead is potent in tumor regression. Our anti-DKK1 lead shows promising efficacy in cancer immunotherapy without influencing Wnt canonical pathway. Citation Format: Linya Wang, Zhen Han, Marisa Yang, Ana G. Lujan Hernandez, Vishwas Prabhu, Cameron F. Hu, Tammy Htoy, Mouna Villalta, Tom Z. Yuan, Hoa Giang, Aaron Sato. Functional DKK1 antibodies binding to cysteine-rich domain 1 or 2 of hDKK1 demonstrate antagonistic activities on tumor suppression or TCF/LEF signaling [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 5793.

Abstract 6345: In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. The leukocyte immunoglobulin (Ig)-like receptor B2 is a member of the leukocyte Ig-like receptor (LILR) family and is predominantly expressed on the surface of cells of the myelomonocytic lineage (monocytes, macrophages and dendritic cells). LILRB2 is a negative regulator of myeloid cells and is an immune checkpoint receptor. We chose to generate mAbs against LILRB2 as a potential immunotherapeutic for cancer. Methods: PentaMice® strains (5 WT mouse strains comprising 9 MHC class II haplotypes to maximize Ab diversity) were immunized with human LILRB2. Lymphocytes were fused with a myeloma partner to generate lead monoclonal hybridomas. 24 purified anti-LILRB2 mAbs were generated and evaluated by arrayed surface plasma resonance (SPR, Carterra® LSA® instrument) to assign epitope bins. mAb humanization was performed in silico to maximize T20 “humanness” scores (algorithm developed by Curia). Humanized mAbs were transiently expressed using Curia’s TunaCHO™ platform and tested in in vitro MDSC assays. Autologous co-cultures of magnetic bead enriched-human blood-derived MDSC (HLA-DR-CD14+) model cells and T cells (CD3+) were stimulated with plate-bound anti-CD3/CD28 to induce T cell activation. Cytokine secretion was assessed 4 days later. Results: A total of 384 hybridoma supernatants were selected from the primary screen. 245 were confirmed by ELISA and 178 stained HEK293 cells expressing LILRB2 cells by flow cytometry (FACS). 24 hybridomas were further selected, cloned and sequenced. Five epitope bins were identified via Carterra® LSA® instrument. Two clones were chosen for humanization, designated 6-I11A and 4-O19A and were recombinantly expressed with the human IgG4 isotype. FACS analysis on HEK293 cells expressing either huLILRB2 or huLILRB4 revealed that clone hu4-O19A-IgG4 was specific for huLILRB2. Surprisingly, clone hu6-I11A-IgG4 stained both huLILRB2 and huLILRB4. FACS analysis with hu4-O19A-IgG4 on human MDSCs exhibited an EC50 of 11.9 nM and a KD of 1.5 nM in binding to recombinant huLILRB2 antigen, while hu6-I11A-IgG4 had an EC50 of 3.2 nM and a KD of 0.6 nM. In co-cultures with human MDSCs and T cells, both hu6-I11A-IgG4 and hu4-O19A-IgG4 reversed cytokine suppression by the MDSCs by increasing GM-CSF, IL-10, TNFα, and IL-6, while suppressing IL-13, and had little effect on IL-17A, MIP-3α, IL-4, IL-2 and IFNγ compared to treatment with the isotype control, with hu6-I11A-IgG4 having a greater effect as an antagonist than hu4-O19A-IgG4. Conclusions: We have identified a dual anti-LILRB2/LILRB4 antagonist. Targeting MDSCs with this antibody offers great promise for immunotherapy for cancer as well as its immense potential for synergy with PD-1 blockade. Citation Format: Bruce L. Daugherty, Xiaomei Ge, Christine L. Hsieh, Brian A. Zabel, Seth Lederman. In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody [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 6345.

Abstract 1859: Diverse CD28-binding IgG and heavy chain-only antibodies for T-cell engager development

Abstract Engagement of the CD28 co-receptor by T-cell engager (TCE) molecules can enhance activation, proliferation, and anti-tumor activities, particularly in immunosuppressive tumor microenvironments. In this study, we present data on a diverse panel of CD28-binding IgG and heavy chain-only (HCAb) antibodies for T-cell co-stimulation. Our diverse panel of CD28-binding antibodies add co-stimulatory building blocks to our TCE repertoire. Using single B cell-screening, we identified fully human IgG and HCAb binders to human and non-human primate CD28 with a high degree of sequence diversity. We selected a subset of antibodies for expression and assessed binding to both human and cynomolgus CD28-expressing cells. These antibodies displayed a wide range of binding avidities in the sub-nanomolar to micromolar range, and epitope binning analysis identified seven epitope communities. Co-stimulatory activity for a subset of molecules was assessed in vitro. Antibodies displayed a wide range of crosslinked T-cell activation with the majority showing no activation independent of crosslinking. Data presented here describe a diverse set of CD28-binding antibodies ready for engineering into co-stimulatory TCEs. Combining these molecules with our TCE platform or other T-cell activating strategies will enable development of novel TCE modalities with co-stimulatory function for diverse tumor targets. Citation Format: Katherine Lam, Shirley Zhi, Lucas Kraft, Elena Vigano, Wei, Cristina Faralla, Esther Odekunle, Kelly Bullock, Erin Marshall, Melissa Cid, Grace Leung, Lindsay DeVorkin, Sherie Duncan. Diverse CD28-binding IgG and heavy chain-only antibodies for T-cell engager development [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 1859.

Abstract 3136: Towards development of a novel biparatopic trop2-ADC

Abstract Background: Trophoblast cell surface antigen 2 (TROP-2) is a member of Tumor-associated Calcium Signal Transducer family (TACSTD2), which is a 35kDa transmembrane protein with 4 N-linked glycosylation sites. The overexpression of TROP2 in various cancers is associated with poor prognosis and increased risk of metastasis. Anti-TROP2 antibody drug conjugates (ADCs) are emerging as an innovative therapeutic intervention for cancer treatment. While these efforts were rewarded by the FDA approval of Trodelvy (Sacituzumab govitecan), a Trop2 targeted ADC, in 2020 for the TNBC patients, tumor heterogeneity could present one of the limitations for the Trop2-targeting therapy, leading to drug resistance. Here, we report our effort in developing a novel biparatopic TROP2 ADC which demonstrate superior cell killing activity compared with the monospecific TROP2 ADC, including certain resistant cell lines for the approved therapy. Methods: Various biparatopic Abs were engineered using two different mAbs targeting the two non-overlapping epitopes of Trop2. The physical stability and biological activities were characterized. Binding competition assay was utilized for epitope binning using SPR. Cell binding affinity was determined using flow cytometry. Cellular internalization activity was measured using Incucyte system, and tumor cell killing efficacy was evaluated in multiple cancer cell lines. CDX mouse models were utilized to investigate the TROP2-ADC in vivo efficacy. Results: We identified a biparatopic TROP2 mAb using two monospecific TROP2 mAbs with A recognizing the same epitope as Trodelvy, and B binding to a non-overlapping epitope vs Sacituzumab’s. mAb-B demonstrates superior ability to recognize a few resistance cell lines to mAb-A. The biparatopic TROP2 mAb-A/B displays a KD of 0.81 nM protein binding compared with 6.42 nM and 0.26 nM, respectively for mAb-A and mAb-B. Importantly, the biparatopic mAb-A/B demonstrates significantly faster cellular internalization activity compared to those of the individual mAbs, signifying the biparatopic scaffold could drive receptor clustering and subsequently enhance the internalization, trafficking to the lysosome and degradation of the target. As expected, the ADC of the biparatopic mAb-A/B conjugated with a TOPi payload shows cancer cell killing efficacy in both in vitro and in vivo experiments. Furthermore, we tested mAb-A, -B and biparatopic mAb-A/B for their binding affinity to 14 missense mutations in TROP2 ECD expressed in HEK293 cells. Interestingly, mAb-A showed decreased binding in 5 mutant cell lines, while mAb-B and biparatopic mAb-A/B maintains activity. Conclusion: We have engineered a novel TROP2 biparatopic antibody with unique biological features as well as superior in vitro and in vivo activities. Such a biparatopic ADC presents its potential to be a best-in-class ADC therapy by overcoming the limitation of the monospecific ADC therapy such as drug resistance. Citation Format: Yinan Wu, Brittany Jiang, Julliet Rivera, Fang Wang, Jing Zhou, Lin Liu, Silvia Hudson, Will Draper, Jennifer Li, Xiaoshan Min, Hang Chen, Zhulun Wang. Towards development of a novel biparatopic trop2-ADC [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 3136.

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Defining the cross-reactivity between peanut allergens Ara h 2 and Ara h 6 using monoclonal antibodies.

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

A tool for analyzing complex epitope binning data of monoclonal antibodies

A tool for analyzing complex epitope binning data of monoclonal antibodies

Discovery of neutralizing SARS-CoV-2 antibodies enriched in a unique antigen specific B cell cluster.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.

A human antibody epitope map of the malaria vaccine antigen Pfs25

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01. These structures revealed additional epitopes in Pfs25 capable of reducing transmission and expanded this characterization to malaria-exposed humans. This work informs immunogen design to focus the antibody response to transmission-reducing epitopes of Pfs25, enabling development of more potent transmission-blocking vaccines for malaria.

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Efficacy of the combination of monoclonal antibodies against the SARS-CoV-2 Beta and Delta variants.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the biggest healthcare issue worldwide. This study aimed to develop a monoclonal antibody against SARS-CoV-2 from B cells of recovered COVID-19 patients, which might have beneficial therapeutic purposes for COVID-19 patients. We successfully generated human monoclonal antibodies (hmAbs) against the receptor binding domain (RBD) protein of SARS-CoV-2 using developed hybridoma technology. The isolated hmAbs against the RBD protein (wild-type) showed high binding activity and neutralized the interaction between the RBD and the cellular receptor angiotensin-converting enzyme 2 (ACE2) protein. Epitope binning and crystallography results displayed target epitopes of these antibodies in distinct regions beneficial in the mix as a cocktail. The 3D2 binds to conserved epitopes among multi-variants. Pseudovirion-based neutralization results revealed that the antibody cocktail, 1D1 and 3D2, showed high potency in multiple variants of SARS-CoV-2 infection. In vivo studies showed the ability of the antibody cocktail treatment (intraperitoneal (i.p.) administration) to reduce viral load (Beta variant) in blood and various tissues. While the antibody cocktail treatment (intranasal (i.n.) administration) could not significantly reduce the viral load in nasal turbinate and lung tissue, it could reduce the viral load in blood, kidney, and brain tissue. These findings revealed that the efficacy of the antibody cocktail, 1D1 and 3D2, should be further studied in animal models in terms of timing of administration, optimal dose, and efficacy to mitigate inflammation in targeted tissue such as nasal turbinate and lung.

Abstract A13: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets. LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the resombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ, whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A13.

Abstract 3185: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets.LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ,whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3185.

Abstract 6366: NB002, a novel therapeutic antibody targeting unique epitope on TIM-3 presenting potent antitumor activity

Abstract Antibody-based Immune checkpoint inhibitor (ICI) therapies targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1) have significantly improved clinical outcomes in cancer treatment. Nonetheless, a large portion of the population has limited benefit from these ICI treatments due to lack of efficacy, resistance and intolerance. T-cell immunoglobulin and mucin domain containing-3 (TIM-3) has been proposed as a target for cancer immunotherapy. It is an inhibitory receptor modulating immune tolerance, and its expression has been broadly reported to be closely correlated to T cell exhaustion and pessimistic prognosis. Besides negatively modulating T cell status, TIM-3 is also expressed on natural killer (NK) cells, dendritic cells (DCs), macrophages and regulates immune responses bridging innate immunity and adaptive immunity. NB002 is a novel humanized Fc-engineered IgG1κ antibody, developed via NeologicsBio’s integrated target validation & antibody screening Tier-A platform. It has been characterized in a serial of function and efficacy studies and completed IND-enabling preclinical development. NB002 binds to human TIM3 with high affinity in the low nanomolar range and antagonize the TIM-3 pathway effectively in reporter cell and primary cell assays. NB002 increased the secretion of IFN-γ by activated PBMC cells and enhanced activation and killing activity of NK cells. Moreover, it exhibited a distinctive activity of restoring myeloid cells and enhancing antigen presentation and processing during anti-tumor immune responses, and superior efficacy in multiple humanized mouse models of cancer as monotherapy regimen. Importantly, X-ray crystallography demonstrated that NB002 recognized a unique epitope on TIM-3 and was confirmed by epitope binning assay and structural alignment. These data could potentially explain novel features of NB002, restoring both acquired and innate immunity which could lead cold tumor to hot tumor.In summary, these data showed that NB002 is a potent anti-TIM-3 antibody with pre-clinical properties. A phase 1 study is planned and patients with advanced metastatic solid tumors will be recruited. Citation Format: Yu Zhang, Jinyu Dong, Binbin Wang, Jie Ni, Dong Wang, Liegang Shao, Meiguang Xiong, Ting Wang, Florencia McAllister, Xin Dong. NB002, a novel therapeutic antibody targeting unique epitope on TIM-3 presenting potent antitumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6366.