Logic-gated antibody pairs that selectively act on cells co-expressing two antigens

The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.


Sta�s�cs
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The exact sample size (n) for each experimental group/condi�on, given as a discrete number and unit of measurement A statement on whether measurements were taken from dis�nct samples or whether the same sample was measured repeatedly The sta�s�cal test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A descrip�on of all covariates tested A descrip�on of any assump�ons or correc�ons, such as tests of normality and adjustment for mul�ple comparisons A full descrip�on of the sta�s�cal parameters including central tendency (e.g. means) or other basic es�mates (e.g. regression coefficient) AND varia�on (e.g. standard devia�on) or associated es�mates of uncertainty (e.g. confidence intervals) For null hypothesis tes�ng, the test sta�s�c (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Data analysis LSR Fortesssa flow cytometry data was processed using FACSDiva v8 and v9.0 so�ware (BD). Sartorius iQue flow cytometry data was processed using iQue ForeCyt v6.2 and v8.1 so�ware (Sartorius). Flow cytometry data were analyzed using FlowJo V10 so�ware (BD). Graphs were plo�ed and analyzed using GraphPad Prism 8.0 (DotMa�cs). EnVision data was processed using EnVision Worksta�on 1.13.3009.1401 so�ware (Perkin Elmer). U-PLEX ProInflam data was processed using MesoScale Diagnos�cs Discovery Workbench v4.0. BioTek absorbance data was analyzed using BioTek Gen5 V1.04.5. BaGoL analysis was performed with the MATLAB implementa�on of BaGoL included with smite (h�ps://github.com/LidkeLab/smite/) version 0.1.0 at the UNM Center for High Performance Compu�ng (CARC) using MATLAB R2019b; post analysis of NN distance per ROI was computed with locally wri�en MATLAB R2019b so�ware using algorithm "knnsearch".
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Sequences of the variable domains and constant domains of recombinant antibodies tested herein were described in patent WO 2019/211472. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Life sciences study design
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Sample size
All data shown are representative of at least three independent replicate experiments or three individual human donors tested. No sample size calculation was performed prior to execution of experiments; post-analysis of experiments demonstrated which group comparisons were sufficiently powered to draw statistically significant conclusions (see Supplementary Tables 3-7). For in vivo studies, group sizes were estimated based on previous experience with similar but not identical experimental settings.
Data exclusions Whole blood donor samples from which insufficient target/effector cells could be recovered were excluded from analysis. All other samples were included. No mice were excluded from analysis, but lipid contamination of some samples precluded FACS analysis.

Replication
All data shown are representative of at least three independent replicate experiments or at least three individual human donors tested. All attempts at replication were successful.
Randomization For in vivo studies, NSG mice were randomly assigned to experimental groups. NSG-HIS mice were randomized into different treatment groups based on the percentage of circulating B and T cells of the total hCD45+ population. For samples used in in vitro studies, randomization was not applicable, as sufficient donor material was available to test each treatment group on each donor sample.

Blinding
For in vivo studies, the investigators who directly performed the study were blinded to the group allocation during execution of the in vivo experiment and the ex vivo analysis. For in vitro studies, group allocation was not applicable, as cells from each donor were exposed to all test items.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. 19. According to statements provided by the manufacturer, antibody S-HCL-1-PE was reactive to human CD22 and validated for use in flow cytometry. 20. According to statements provided by the manufacturer, antibody HIB19-PE-CF594 was reactive to human CD19 and validated for use in flow cytometry. 21. According to statements provided by the manufacturer, antibody RPA-2.10-APC was reactive to human CD2 and validated for use in flow cytometry. 22. According to statements provided by the manufacturer, antibody RmC11H9-FITC was reactive to mouse C3 and validated for use in flow cytometry. 23. According to statements provided by the manufacturer, antibody 2.4G2 was reactive to mouse Fc and validated for use in flow cytometry. 24. According to statements provided by the manufacturer, antibody Fc1.3216 was reactive to human Fc and validated for use in flow cytometry. 25. According to statements provided by the manufacturer, antibody MφP-9-FITC was reactive to human CD14 and validated for use in flow cytometry. 26. According to statements provided by the manufacturer, antibody J3-119-PE was reactive to human CD19 and validated for use in flow cytometry. 27. According to statements provided by the manufacturer, antibody Bu15-PE-Cy7 was reactive to human CD11c and validated for use in flow cytometry. 28. According to statements provided by the manufacturer, antibody WM53-APC was reactive to human CD33 and validated for use in flow cytometry. 29. According to statements provided by the manufacturer, antibody CMSSB-PerCP-eFluor710 was reactive to human CD56 and validated for use in flow cytometry. 30. According to statements provided by the manufacturer, antibody 30-F11-APC-eFluor780 was reactive to mouse CD45 and validated for use in flow cytometry. 31. According to statements provided by the manufacturer, antibody NB600-363AF647 was reactive to the HA tag and validated for use in immunofluorescence.

Wild animals
The study did not involve wild animals.
Field-collected samples The study did not involve field-collected samples. Note that full information on the approval of the study protocol must also be provided in the manuscript.

nature research | reporting summary
April 2020

Human research participants
Policy information about studies involving human research participants

Population characteristics
Population characteristics of blood samples or derivatives from healthy human donors were blinded to the authors in accordance with GDPR policy. Population characteristics of PBMCs derived from CLL patients are described in Supplementary  Table 9. Importantly, this study did not involve any diagnostics specific to age, gender, or race, or draw any conclusions based on these patient characteristics.

Recruitment
PBMCs derived from CLL patients were commercially obtained from Discovery Life Sciences (Huntsville, AL, USA). Buffy coats from healthy human donors and complement-competent, pooled normal human serum (NHS; AB positive) were obtained from Sanquin (Amsterdam, The Netherlands). Whole blood samples from healthy human volunteers were freshly obtained from the University Medical Center Utrecht (Netherlands).

Ethics oversight
Commercially available patient-derived PBMCs and healthy donor blood(-derived) samples were collected at the site of the vendor after patient written and informed consent in accordance with the declaration of Helsinki. All vendors maintained strict ethical compliance, including fully de-identified materials and stringent Institutional Review Board (IRB) and Ethics Committee compliance.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
For an in-depth description of sample preparation, we refer to the individual assay sections within the online methods: cells and reagents, whole blood cytotoxicity, C1q binding, CDC, ADCP, FRET, and In Vivo POC studies.
Cell population abundance N.A., as no cell sorting was performed.

Gating strategy
After gating of forward scatter (FSC) vs. side scatter (SSC), doublets were excluded by FSC-A vs. FSC-H. For whole blood assays, leukocytes were selected by CD45+ staining. Dead cells were excluded by fixable viability stain (FVS). Lymphocytes and granulocytes were separated based on CD66b staining. B-and T cells within the lymphocyte (CD66b-) cell population were identified as CD19+ and CD3+/(CD4+), respectively. More details are supplied in supplementary table 1 and  supplementary figure 7. For ADCP assays, target cells were distinguished from hMDM by CD11b, CalceinAM and CD19 staining as described in the 'ADCP' paragraph in the methods section. For binding assays, binding was quantified as the geometric mean fluorescent intensity (gMFI) of a directly-labeled antibody, or fluorochrome-conjugated secondary antibody respectively. More details are supplied in supplementary table 2  Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.