Antibody Constructs Research Articles

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens.

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Abstract 2891: Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody

Abstract Intracellular tumor antigens presented as peptides on MHC (pMHC) class I molecules are attractive targets for more tumor-selective immunotherapeutic approaches with promising data already emerging from clinical trials. pMHCs have been targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment. Naturally occurring cancer-reactive TCRs have weak affinity and require substantial affinity enhancements for their cognate pMHC. However, the outcome of this process is difficult to predict and bears the risk for off-target cross reactivities in normal tissues, which may lead to severe adverse events in the clinic. Here, we describe a highly potent dual pMHC T-cell engager (TCE) antibody format with high specificity towards tumor-specific pMHCs utilizing an HLA-A*02:01 restricted MAGE-A4 epitope to validate the concept. A series of monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding arms, ranging in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment with lower affinity compared to that commonly used for TCR-fusions. The different antibody constructs were evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS osteosarcoma and A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-A*02-positive human cell lines. Bivalent bispecific antibody variants mediated a 7-fold greater degree of cancer cell killing and similarly increased T cell activation compared to their monovalent bispecific counterparts. EC50 values ranged as low as single digit picomolar, while the overall cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not substantially affected. These results prove that dual targeting of pMHCs on cancer cells provides selective and efficient T cell-mediated target cell killing and T cell activation, even at very low levels of pMHC on the cell surface, highlighting the pivotal roles played by the affinity of the individual arms, valency, and epitope densities. We further analyzed the dual pMHC TCE for potential off-target effects by recognition of similar and physiologically relevant non-MAGE-A4 peptides. T2 cells pulsed with similar peptides and co-cultured with PBMC effector cells showed no significant T cell activation or IFNg release in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed T2 cells. Finally, we compared the dual pMHC TCE against a recombinant TCR fused to an anti-CD3 scFv, a construct that is currently in clinical development. Interestingly, the dual pMHC TCE resulted in a 3-fold more potent cancer cell killing while having significantly lower effect on cytokine production. In conclusion, dual pMHC targeting with TCE antibodies is an attractive alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as they facilitate potent tumor targeting without the need for extensive affinity enhancements. Citation Format: Stephanie Jungmichel, Fabian Scheifele, Anna Sobieraj, Severin Wendelspiess, Thomas Schleier, Philip Knobel, Camilla Winnewisser, Melissa Vrohlings, Philipp Richle, Hannes Merten, Jacqueline Blunschi, Christian Leisner, Leonardo Borras. Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2891.

Abstract 2898: HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC)

Abstract Background: Advanced prostate cancers (PC) are largely insensitive to existing immunotherapies. T-cell-engaging antibodies, linking tumor cells to T cells, are being developed to treat malignancies. HPN424 is a Tri-specific T-cell Activating Construct (TriTAC), binding PSMA, CD3ε, and albumin to improve drug half-life. We evaluated the activity of HPN424 to induce T-cell-mediated cytotoxicity against organoids generated from patient-derived xenograft (PDX-Os) established from human mCRPC biopsies. Design: PDX-Os from three models were evaluated; their membranous PSMA immunohistochemical expression was quantified using H-scores (HS). PDX-Os were co-cultured with: i) healthy donor allogeneic T cells, ii) healthy donor allogeneic T cells combined with HPN424 (1pM, 10pM or 100pM), or iii) healthy donor allogeneic T cells combined with PL711, a non-targeting TriTAC control (1pM, 10pM, 100 pM). Cytotoxicity was determined by LDH release and visualisation of T cell/PDO co-localisation and PDO disruption using confocal microscopy. Early and mid-late T cell activation was determined by FACS for CD69 and CD25 expression, respectively. Cytolytic (CTL) and CD4 T cell cytokine release (IFNγ, TNF∝, IL-1β/2/4/6/8/10) was evaluated using the AlphaLISA kit (MSD). FACS studies were performed after 48- and 72-hours, while microscopy, LDH and cytokine assays were performed after 24, 48, and 72-hours, of co-culture respectively. Results: In the highest PSMA-expressing PDX-Os (CP50c, HS: 225; CP142c, HS: 165), co-culture of resting healthy donor T cells with CRPC PDX-Os led to dose-dependent increases in LDH release at 48-hours (p<0.0001) and 72-hours (p<0.0001), in the presence of HPN424 but not PL711. Correspondingly, in these models, confocal microscopy showed T cells co-localising with and disrupting the PDX-Os by 24-hours across all 3 HPN424 concentrations tested. HPN424 induced T-cell expression of CD25 and CD69 within 48-hours of co-culture across all 3 concentrations in both models. When compared with PL711, HPN424 induced dose-dependent upregulation of IFNγ, IL-1β, IL-2, IL-4, IL-10, and TNF∝ in the PSMA-high PDX-Os, CP50c and CP142c (all p<0.0001), indicating T cell activation and induction of CTL and Th1/Th2/Treg cytokines by HPN424. Interestingly, in co-cultures of T cells and CP89c PDX-Os, which have lower PSMA expression (HS of 25), HPN424 did not significantly increase LDH release when compared with PL711; in this model CD25/CD69 induction and the upregulation of IFNγ, IL-1β/2, and TNF∝ was only observed at the highest concentration of HPN424 (100pM). Conclusions: HPN424, a PSMA-targeting TriTAC antibody construct, induced T cell activation and elicited T-cell mediated cytotoxicity against PSMA expressing mCRPC PDX-Os. Our data support further evaluation of this agent in advanced PC with membranous PSMA expression. Citation Format: Veronica Gil, Christina Guo, George Seed, Antje Neeb, Lorenzo Buroni, Bora Gurel, Ines Figueiredo, Golzar Hemmati, Wade Aaron, Johann S. de Bono. HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2898.

Abstract 2078: CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells

Abstract Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with few options for curative treatment. In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable success. However, such immunotherapeutic strategies are not yet established for AML, likely due to a dearth of appropriate targets. The fms like tyrosine kinase 3 (FLT3)/CD135 surface receptor is expressed on AML cells in the large majority of AML patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. We report here on the development of CLN-049, a bispecific humanized antibody that binds to FLT3 on leukemic cells and the CD3 epsilon subunit of the T cell receptor complex on T cells. CLN-049 recognizes the membrane proximal domain of FLT3 and has a silenced Fc gamma 1 domain, with anti-CD3 single chain variable fragments (scFv) fused to the C-termini of the heavy chains. While FLT3 kinase inhibitors are limited to treatment of patients with specific intracellular FLT3 mutations, CLN-049 recognizes the extracellular domain of FLT3 and would therefore be more broadly applicable in AML therapy. CLN-049 showed low-nanomolar binding to both FLT3-expressing and CD3-expressing cells. The antibody induced target-dependent activation of CD4+ and CD8+ T cells as well as corresponding cytokine production by T cells only when co-cultured with FLT3+ AML cells. Human AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed upon treatment with sub-nanomolar concentrations of CLN-049 when co-cultured with heterologous PBMCs, regardless of the expression level or mutational status of FLT3. Intriguingly, FLT3-expressing hematopoietic progenitor cells and dendritic cells were not found to be sensitive to CLN-049 killing. The in vitro lysis of leukemic cells by CLN-049 was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft studies, CLN-049 was highly active against the MOLM-13 human AML model. In these studies, CLN-049 demonstrated significant dose-dependent activity as measured by survival as well as reduction of AML cells in the blood. In summary, CLN-049 is a promising FLT3-targeted T cell engaging antibody construct expected to have robust anti-tumor activity in the clinic against AML. CLN-049 is currently in a phase 1 clinical trial for the treatment of patients with relapsed/refractory AML. Citation Format: Kristan Meetze, Naveen K. Mehta, Martin Pfluegler, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson, Gundram Jung, Helmut Salih. CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2078.

Abstract CT003: Innate cell engager (ICE®) AFM13 combined with preactivated and expanded cord blood (CB)-derived NK cells for patients with refractory/relapsed CD30+ lymphoma

Abstract Pts with R/R CD30+ lymphoma have few effective therapies available. We previously developed a promising therapy using CB-derived NK that were first IL-12/IL-15/IL-18-preactivated followed by expansion (P+E) with K562 feeder cells ex vivo and subsequently complexed with ICE® AFM13 (a first-in-class CD30/CD16A bispecific antibody construct) prior to infusion. These cytokine-induced memory-like NK showed greater in vivo antitumor activity than either P+E NK or AFM13 alone. This single-center phase I-II trial (NCT04074746) evaluates AFM13-precomplexed CB-NK cells in pts ages 15-75 with R/R CD30+ lymphoma. Pts receive 2 cycles of fludarabine/cyclophosphamide (days −5 to −3) followed by AFM13-CB NK cells (day 0) and 3 weekly intravenous infusions of AFM13 (200 mg, days 7, 14 and 21). Pts were enrolled at 3 dose levels: DL1 (106 NK/Kg), DL2 (107 NK/Kg) and DL3 (108 NK/Kg). 19 pts have been treated to date at DL1 (N=3), DL2 (N=3) and DL3 (N=13); 18 have completed both planned cycles. All pts had active progressive disease at enrollment and no bridging therapy was given. Cords were selected for the 37 cycles without consideration for HLA match, which was 0/6 (N=16), 1/6 (N=17), 2/6 (N=2), 3/6 (N=1) and 4/6 (N=1). There were no cases of CRS, ICANS or GVHD; 6 infusion-related reactions (1 G3, 5 G2) in 110 infusions of AFM13 alone and no reactions to the AFM13-loaded NK cells. DL3 was established as the RP2D. There were 17/19 responses (8 CR, 9 PR, 2 PD). All 13 pts treated at the RP2D responded (6 CR, 7 PR). 2 pts subsequently received an autologous SCT. At median follow-up of 6 (2-16) months PFS/OS across all dose levels are 58%/79%. Expansion of CB NK cells occurred as early as 3 days post infusion and persisted for up to a month. In conclusion, the preliminary results of this first clinical trial of ICE®-precomplexed NK cells for R/R CD30+ lymphoma indicate excellent tolerability and high activity and warrant further investigation of this approach. Baseline patient characteristics (N=19) Age, median (range) 37 (20-68) Gender (male/female) 13/6 Diagnosis (Hodgkin/T-NHL) 17/2 No. prior lines therapy, median (range) 6 (1-14) Prior brentuximab vedotin 19 Prior anti-PD-1 18 Prior SCT (autologous/allogeneic) 13 (8/5) Prior cellular therapy (CAR-T) 2 No. prior relapses/progressive disease, median (range 5 (1-14) Citation Format: Yago Nieto, Pinaki Banerjee, Indreshpal Kaur, Roland Bassett, Lucila Kerbauy, Rafet Basar, Mecit Kaplan, Lori Griffin, Daniel Esqueda, Christina Ganesh, Melissa Barnett, Amin Alousi, Chitra Hosing, Jeremy Ramdial, Neeraj Saini, Samer Srour, Karenza Alexis, Andreas Harstrick, Elizabeth J. Shpall, Katayoun Rezvani. Innate cell engager (ICE®) AFM13 combined with preactivated and expanded cord blood (CB)-derived NK cells for patients with refractory/relapsed CD30+ lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT003.

Abstract 2077: Preclinical characterization of a next-generation CD19/CD3-bispecific T cell engager with extended serum half-life and superior potency against CD19-low target cells

Abstract T cell therapies targeting the B cell antigen CD19 have profoundly changed the way we treat various B cell malignancies. Both the CD19-specific T cell-engaging bispecific antibody blinatumomab and various autologous CAR-T cell therapies have exhibited robust clinical activity in relapsed/refractory acute lymphocytic leukemia (ALL) and non-Hodgkin lymphoma (NHL). While CAR-T cell therapies show high complete response rates, bispecific antibodies have room for improvement. Both therapies, however, fail in patients where tumor cells escape selective pressure by expressing very low or undetectable levels of the CD19 antigen.We developed a novel CD19/CD3-bispecific antibody construct called CLN-978. Like blinatumomab, CLN-978 has tandemly arranged CD19- and CD3-specific single-chain Fv (scFv) fragments. Uniquely, a single-domain antibody specific for serum albumin was N-terminally fused to enable a prolonged serum half-life, eliminating the need for continuous IV infusion. CLN-978 is composed of novel humanized CD19-, CD3- and albumin-binding elements that are cross-reactive with non-human primate (NHP) orthologs and lacks a hexahistidine tag. It is produced at g/L levels in CHO cells and enables protein A affinity purification. CLN-978 has a 100-fold higher binding affinity for CD19 than blinatumomab. Side-by-side comparison with blinatumomab showed that the higher CD19 binding affinity translates into superior T cell-mediated cytotoxicity (TDCC) of CLN-978 with both target cell lines expressing high or very low levels of CD19. Superior efficacy was similarly observed in xenotransplant mouse tumor models. Preclinical studies in primates are in progress assessing safety, pharmacology, pharmacokinetics (PK) and pharmacodynamics (PD) of CLN-978 administered either through intravenous or subcutaneous delivery. CLN-978 has the potential to have superior TDCC against cells expressing very low levels of CD19 as are selected following treatment with CD19-directed therapies. This may translate into higher response rates and longer response duration in ALL and NHL patients expressing normal and very low levels of CD19 on malignant B cells. The cross-reactivity of CLN-978 to the NHP orthologs of CD19, CD3 and albumin enables assessment of depletion of normal B cells, strategies to mitigate cytokine release, PK and PD properties, and exploration of dosing routes. CLN-978 holds promise as a CD19-targeted therapeutic that can be easily and conveniently administered to patients with diverse B cell malignancies. Citation Format: Patrick A. Baeuerle, Naveen K. Mehta, Kristan Meetze, Bochong Li, Jennifer S. Michaelson. Preclinical characterization of a next-generation CD19/CD3-bispecific T cell engager with extended serum half-life and superior potency against CD19-low target cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2077.

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.

Passive Immunotherapy Against SARS-CoV-2: From Plasma-Based Therapy to Single Potent Antibodies in the Race to Stay Ahead of the Variants.

The COVID-19 pandemic is now approaching 2 years old, with more than 440 million people infected and nearly six million dead worldwide, making it the most significant pandemic since the 1918 influenza pandemic. The severity and significance of SARS-CoV-2 was recognized immediately upon discovery, leading to innumerable companies and institutes designing and generating vaccines and therapeutic antibodies literally as soon as recombinant SARS-CoV-2 spike protein sequence was available. Within months of the pandemic start, several antibodies had been generated, tested, and moved into clinical trials, including Eli Lilly’s bamlanivimab and etesevimab, Regeneron’s mixture of imdevimab and casirivimab, Vir’s sotrovimab, Celltrion’s regdanvimab, and Lilly’s bebtelovimab. These antibodies all have now received at least Emergency Use Authorizations (EUAs) and some have received full approval in select countries. To date, more than three dozen antibodies or antibody combinations have been forwarded into clinical trials. These antibodies to SARS-CoV-2 all target the receptor-binding domain (RBD), with some blocking the ability of the RBD to bind human ACE2, while others bind core regions of the RBD to modulate spike stability or ability to fuse to host cell membranes. While these antibodies were being discovered and developed, new variants of SARS-CoV-2 have cropped up in real time, altering the antibody landscape on a moving basis. Over the past year, the search has widened to find antibodies capable of neutralizing the wide array of variants that have arisen, including Alpha, Beta, Gamma, Delta, and Omicron. The recent rise and dominance of the Omicron family of variants, including the rather disparate BA.1 and BA.2 variants, demonstrate the need to continue to find new approaches to neutralize the rapidly evolving SARS-CoV-2 virus. This review highlights both convalescent plasma- and polyclonal antibody-based approaches as well as the top approximately 50 antibodies to SARS-CoV-2, their epitopes, their ability to bind to SARS-CoV-2 variants, and how they are delivered. New approaches to antibody constructs, including single domain antibodies, bispecific antibodies, IgA- and IgM-based antibodies, and modified ACE2-Fc fusion proteins, are also described. Finally, antibodies being developed for palliative care of COVID-19 disease, including the ramifications of cytokine release syndrome (CRS) and acute respiratory distress syndrome (ARDS), are described.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40259-022-00529-7.

Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy.

Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a therapeutic target. In order to examine the use of TK1 as a therapeutic target, it is necessary to develop therapeutics specific for it. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had an estimated limit of detection of 3.9 ng/ml for the antibody fragments 4-H-TK1_A1 and 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mononuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNCs, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti-TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 on tumor cells.

212Pb-conjugated anti-rat HER2/neu antibody against a neu-N derived murine mammary carcinoma cell line: cell kill and RBE in vitro

Purpose In the current work, the RBE of a 212Pb-conjugated anti-HER2/neu antibody construct has been evaluated, in vitro, by colony formation assay. The RBE was estimated by comparing two absorbed dose-survival curves: the first obtained from the conjugated 212Pb experiments (test radiation), the second obtained by parallel experiments of single bolus irradiation of external beam (reference radiation). Materials and Methods Mammary carcinoma NT2.5 cells were treated with (0–2.47) kBq/ml of radiolabeled antibody. Nonspecific binding was assessed with addition of excess amount of unlabeled antibody. The colony formation curves were converted from activity concentration to cell nucleus absorbed dose by simulating the decay and transport of all daughter and secondary particles of 212Pb, using the Monte Carlo code GEANT 4. Results The radiolabeled antibody yielded an RBE of 11.5 at 37% survival and a survival independent RBE (i.e. RBE2) of 13.7. Unbound/untargeted 212Pb-labeled antibody, as obtained in blocking experiments yielded minimal alpha-particle radiation to cells. Conclusions: These results further highlight the importance of specific targeting toward achieving tumor cell kill and low toxicity to normal tissue.

Blinatumomab as a Bridge Therapy for Hematopoietic Stem Cell Transplantation in Pediatric Refractory/Relapsed Acute Lymphoblastic Leukemia.

Simple SummaryImmunotherapies are modern treatment modalities, giving hope for improvements of frozen cure rates in many childhood malignancies. More intensive cytotoxic chemotherapy cycles didn’t improve cure rates, only increase number of adverse events. Blinatumomab, a bispecific CD3/CD19 antibody construct, has been successfully used in relapsed/refractory r/r B-cell precursor ALL (BCP-ALL) as a bridge to hematopoietic stem cell transplantation (HSCT). We retrospectively assessed the efficacy and toxicity of blinatumomab in 13 children with r/r BCP-ALL. The response rate in our cohort of patients was 85%, with subsequent feasible HSCT in 11 out of 13 children. Although our study had some limitations with regard to its retrospective design and limited patient population, it clearly showed blinatumomab as not only a feasible but also an effective therapeutic option in pretreated children with r/r BCP-ALL, with a tolerable toxicity profile, paving the way for an HSCT procedure. To date, this is the first retrospective study from Poland on efficacy and toxicity of blinatumomab therapy in children with r/r ALL.Despite the progress that has been made in recent decades in the treatment of pediatric acute leukemias, e.g., converting acute lymphoblastic leukemia (ALL) from a fatal to a highly curable disease, 15–20% of children still relapse. Blinatumomab, a bispecific CD3/CD19 antibody construct, has been successfully used in relapsed/refractory r/r B-cell precursor ALL (BCP-ALL) as a bridge to hematopoietic stem cell transplantation (HSCT). We retrospectively assessed the efficacy and toxicity of blinatumomab in 13 children with r/r BCP-ALL. Between 2017 and 2021, thirteen children, aged 1–18 years, with r/r BCP-ALL were treated with blinatumomab. Two patients were administered blinatumomab for refractory relapse without complete remission (CR), one due to primary refractory disease, and ten patients were in CR with minimal residual disease (MRD) ≥ 10−3. The response rate in our cohort of patients was 85%, with subsequent feasible HSCT in 11 out of 13 children. Ten children reached MRD negativity after the first blinatumomab administration. The three-year OS for the study patients was 85% (Mantel–Cox, p < 0.001) and median follow-up was 24.5 (range: 1–47). All responders proceeded to HSCT and are alive in CR, and MRD negative. Although our study had some limitations with regard to its retrospective design and limited patient population, it clearly showed blinatumomab as not only a feasible but also an effective therapeutic option in pretreated children with r/r BCP-ALL, with a tolerable toxicity profile, paving the way for an HSCT procedure.

Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates &amp;gt;50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

Trial in Progress: A Phase 2 Single Arm, Multicenter Trial to Evaluate the Efficacy of the BiTE ® Antibody Blinatumomab (Blincyto) and Vincristine Sulfate Liposomal Injection (Marqibo) in Adult Subjects with Relapsed/Refractory Philadelphia Negative CD19+ Acute Lymphoblastic Leukemia

Trial in Progress: A Phase 2 Single Arm, Multicenter Trial to Evaluate the Efficacy of the BiTE ® Antibody Blinatumomab (Blincyto) and Vincristine Sulfate Liposomal Injection (Marqibo) in Adult Subjects with Relapsed/Refractory Philadelphia Negative CD19+ Acute Lymphoblastic Leukemia

Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML.In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases).We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D).Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. [Display omitted] DisclosuresRadojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Blinatumomab for Patients with Richter's Syndrome: A Multicenter Phase 2 Trial from the Filo Group

BackgroundRichter syndrome (RS) refers to the onset of aggressive lymphoma, mostly diffuse large B cell lymphoma (DLBCL), in patients with chronic lymphocytic leukemia (CLL). The outcome of RS patients is usually very poor with both low response rates to chemoimmunotherapy and short survival. While BCR and BCL2 inhibitors have transformed the management of CLL patients, these drugs do not prevent the onset of RS. Modulating anti-tumor immunity has recently been suggested as a promising approach in RS (Ding, 2017).Blinatumomab is a bi-specific T-cell engaging antibody construct that transiently links CD3-positive T cells to CD19-positive B-cells, inducing T-cell activation and subsequent lysis of tumor cells. It has been approved for the treatment of patients with relapsed or refractory B-ALL and has also been evaluated in the setting of persisting minimal residual disease. Recently, blinatumomab (stepwise dosing 9-28-112 μg/d) has been evaluated in patients with relapsed or refractory DLBCL and demonstrated promising results (ORR 43%) with acceptable safety (Viardot, 2016).We hypothesized that blinatumomab would improve response in RS patients failing to achieve CR after initial debulking with R-CHOP.MethodsWe report here the first results of a phase 2 multicenter study investigating the efficacy and safety of blinatumomab after R-CHOP debulking therapy for patients with untreated RS of DLBCL histology (NCT03931642). The patients with persisting (PR, SD) or progressive disease (PD) after 2 cycles of R-CHOP were eligible to receive an 8-week course of blinatumomab induction. Blinatumomab was administered at a stepwise dose of 9 μg/d in the first week, 28 μg/d in the second week, and 112 μg/d thereafter. The primary endpoint was CR rate according to the revised Lugano criteria after the 8-week induction course of blinatumomab. An additional 4-week consolidation cycle was optional. Allo-HSCT was further allowed for eligible patients.ResultsA total of 34 patients out of 41 has already been enrolled in the trial to date. Median age was 66 years (range, 38-82) and sex ratio M/F was 23/12. CLL features at baseline were as follows: 57% had 17p deletion and 67% TP53 mutations. Sixty-five percent had complex karyotype and 79% unmutated IGHV status. Median number of prior therapeutic lines for CLL was 2 (range, 0-7): 19 (54%) patients previously received chemo-immunotherapy, 23 (66%) patients were exposed to ibrutinib and 11 (31%) to venetoclax.As of the data cut-off of June 1st, 2021, the blinatumomab induction course has been completed for 18 patients. Ten patients did not receive blinatumomab for the following reasons: 7 patients achieved CR after R-CHOP, 2 patients died because of febrile neutropenia after R-CHOP and 1 patient presented severe pneumonia after R-CHOP. Three patients are still on R-CHOP and 3 others on blinatumomab to date.Regarding toxicity during blinatumomab, data are available for the 18 patients having completed the blinatumomab induction to date. All patients had at least one grade 1 adverse event (AE), 10 had grade ≥3 AE. The most common AE (> 1 case), regardless of relationship to blinatumomab, were fever (4 patients), CRS (2 patients), sepsis (2 patients), vein thrombosis (2 patients), anemia (4 patients), neutropenia (3 patients), lymphopenia (5 patients), thrombocytopenia (3 patients) and hyperglycemia (5 patients). In terms of neurologic events, 5 (28%) experienced neurotoxicity (all recovered) including grade 3 encephalopathy, grade 4 confusion, grade 3 anxiety, grade 1 myoclonus, grade 2 ataxia, grade 1 sleep disorder and grade 1 ICANS (each in 1 pt). Blinatumomab was temporarly stopped in 3 patients and permanently in 2.In terms of efficacy, after R-CHOP debulking therapy (n=31 evaluable patients), 7 patients achieved CR, 6 patients were in PR, 7 patients were stable and 11 patients were progressive. At evaluation after the blinatumomab induction (n=18 evaluable patients), 4 (22.2%) patients achieved CR, 4 (22.2%) patients PR, 2 (11.1%) patients were stable and the remaining 8 (44.5%) were progressive. Considering the whole strategy (including R-CHOP debulking) (n=28), 15 (54%) patients achieved overall response including 11 (39%) CR.ConclusionsOur preliminary data suggest that blinatumomab suggests encouraging anti-tumor activity and acceptable toxicity in patients with RS. DisclosuresYsebaert: Abbvie, AstraZeneca, Janssen, Roche: Other: Advisory Board, Research Funding. Ferrant: AstraZeneca: Honoraria; Janssen: Other: Travel, Accommodations, Expenses; AbbVie: Honoraria, Other: Travel, Accommodations, Expenses. de Guibert: Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Laribi: Astellas Phama, Inc.: Other: Personal Fees; AstraZeneca: Other: Personal Fees; Novartis: Other: Personal Fees, Research Funding; Le Mans Hospital: Research Funding; IQONE: Other: Personal Fees; Jansen: Research Funding; BeiGene: Other: Personal Fees; Takeda: Other: Personal Fees, Research Funding; AbbVie: Other: Personal Fees, Research Funding. Feugier: Abbvie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Honoraria; Astrazeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. OffLabel Disclosure:blinatumomab is approved for acute lymphoblastic leukemia. The aim of this phase 2 study is to evaluated it in patients with Richter's syndrome.

A Bispecific Antibody Targeting CD117 and CD3 Enables T Cell Mediated Killing of CD117-Expressing Healthy and Malignant Hematopoietic Cells

INTRODUCTIONHematopoietic stem and progenitor cells (HSPCs) support life-long hematopoiesis. A single HSPC can also be at the origin of hematological malignancies, such as Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). Allogeneic HSCT with the intent to eliminate recipient AML or MDS and at the same time replace recipient HSPC with donor-HSPC and immune cells is a life-saving therapeutic option for many patients. However, chemotherapy (and sometimes in addition gamma-irradiation based conditioning regiments) prior to HSCT are associated with substantial toxicity. Thus, due to benefit-outweighing treatment-related toxicity and mortality, frail, multi-morbid and elderly patients are usually excluded from potentially curative allo-HSCT approaches. For these reasons, more selective preconditioning strategies, leading to residual AML/MDS elimination and creating “space” for incoming HSPCs, are required. Selective targeting of CD117 with monoclonal antibodies has been proposed as a strategy to remove endogenous HSPCs, enabling an effective but mild preconditioning. However, specific conditioning of AML and MDS patients, prior to HSCT, might require a more potent effector cell type. We hypothesized that a CD117 and CD3 binding, T cell engaging and activating antibody construct (CD117xCD3 TEA) with a short half-life might be an ideal means to selectively eliminate CD117-expressing healthy HSPCs and residual CD117-expressing AML or MDS cells prior to allo-HSCT. METHODSWe cloned and expressed CD117xCD3 TEA in tandem scFv format and produced it by transient gene expression in Chinese hamster ovary cells (CHO-S). The fusion proteins were purified to homogeneity by protein A affinity chromatography. We derived target cell lines with varying surface levels of CD117 (high, medium and low) from CD117 negative parental cell lines HL-60 and MOLM-14 (Myburgh et al., Leukemia, 2020). To assess T cell mediated killing of target cells, we mixed them with human T cells (purified and enriched after negative selection) at varying Effector-to-Target (E:T) cell ratios and added CD117xCD3 TEA at different concentrations. The mixture was incubated and specific killing was quantified via flow cytometry at different time-points. RESULTSIn order to characterize the biocidal properties of CD117xCD3 TEA, we performed in vitro killing experiments against cell lines, HSCPs from healthy donors and blast cells from AML patients. A dose-dependent in vitro killing of the cell lines was observed in the presence of various concentrations of CD117xCD3 TEA and of human T cells at an E:T cell ratio of 10:1 after 24h. The HL60 CD117 high cell line was efficiently lysed (~90%) at 100 ng/ml of CD117xCD3 TEA, corresponding to ~1.8 nM. In similar experiments with different E:T cell ratios, we observed that both HL60 CD117 high and CD117 medium cells could be quantitatively killed at E:T ratios as low as 1:1, while the killing of CD117 low cells required a higher density of T cells. The biocidal effect on non-transduced HL60 cells was negligibly low, confirming the requirement of a simultaneous engagement of CD117 and CD3 for specific killing. We repeated the same experiment with an engineered MOLM14 cell line, which also expressed CD117 at comparable high levels, incubating the target cell line with human T cells at an E:T of 1:1 for 24, 48 or 72, 120 or 192 hours. Complete killing of the target cell line was achieved at 120 and 192 hours and after supplemental addition of T cells and CD117xCD3 TEA at 72 hours (see example figure). Experiments with primary cells (HSPCs from healthy donors or blast cells from AML patients) at an E:T of 1:1 confirmed specific killing of target cells in an antigen-density- and concentration-dependent manner after 48h. CONCLUSIONSWe have generated a novel bispecific antibody, which binds to human CD117 (expressed on HSCPs and AML/MDS blast cells) and to CD3 (expressed on T cells), which we term CD117xCD3 TEA. The antibody induces selective T cell-mediated killing of cell lines with different surface levels of CD117, as well as of healthy HSPCs and primary human AML cells. Thus, the newly generated CD117xCD3 TEA might be developed clinically in order to erradicate residual AML/MDS and at the same time serve as a milder preconditioning approach prior to allo-HSCT in frail AML/MDS patients. [Display omitted] DisclosuresKiefer: ETH Zurich: Current Employment, Patents & Royalties: CD117xCD3 TEA. Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Guggisberg: F. Hoffmann-La Roche AG: Current Employment. Abdelmotaleb: F. Hoffmann-La Roche AG: Current Employment. Mock: Philogen S.p.A.: Current Employment. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: University of Zurich: Patents & Royalties: CD117xCD3 TEA; CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company.

Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses.

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen.

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.