The authors have no acknowledgements.

1. Evaluation of Target Binding by Flow Cytometry
<ol>
	<li>Preparation of biological materials and reagents
	<ol>
		<li>Make 500 mL of RPMI culture medium supplemented with 10% heat-inactivated fetal bovine serum (H-IFBS).</li>
		<li>Culture Daudi Burkitt&#39;s Lymphoma (Daudi) cells and Daudi GFP+ cells using RPMI and 75-cm2 culture flasks. Maintain the cultures at 37 &#176;C in a 5% CO2 humidified atmosphere until they reach 6 - 9 x 105 cells/mL.</li>
		<li>Make 50 mL of staining buffer by diluting 1/100 H-IFBS in PBS; this buffer is stable at 2 - 8 &#176;C for at least one month.</li>
		<li>Prepare the test solutions for the reference and biosimilar mAbs. Make ten 1:2 serial dilutions (500 &#181;L each) in staining buffer, starting from 5 &#181;g/mL.</li>
		<li>Use staining buffer to dilute human IgG (isotype control) to 5 &#181;g/mL and PE-Cy5 mouse anti-human IgG (secondary antibody) to the concentration suggested by the manufacturer.</li>
		<li>Prepare 4% paraformaldehyde in PBS (fixation buffer).</li>
	</ol>
	</li>
	<li>Target binding
	<ol>
		<li>Collect the Daudi and Daudi GFP+ cell suspensions from the 75-cm2 culture flasks and transfer them to a 15-mL centrifuge tube. Centrifuge at 400 x g for 5 min.</li>
		<li>Wash the cells by adding 5 mL of PBS and centrifuging the cell suspension at 400 x g for 5 min.</li>
		<li>Resuspend the cells in PBS and perform a cell count and viability analysis with trypan blue. Use cultures with cell viability levels &#8805; 95% for the analysis.</li>
		<li>Dilute the cell suspension to 4 x 106 cells/mL with cold staining buffer.</li>
		<li>In 1.5-mL microcentrifuge tubes, add 50 &#181;L of the cell suspension to 100 &#181;L of the different test concentrations of the reference or biosimilar mAbs. Include replicates for each experimental condition.</li>
		<li>Prepare additional tubes for the isotype control (human IgG1 instead of rituximab) and negative control (secondary antibody without primary antibody).</li>
		<li>Incubate at 4 &#176;C for 20 - 30 min.</li>
		<li>Wash the cells by adding 1 mL of PBS and centrifuging the cell suspension at 400 x g for 5 min at 10 &#176;C. Discard the supernatant.</li>
		<li>Suspend the cells in 100 &#181;L of the secondary antibody and incubate for 20 - 30 min at 4 &#176;C, protected from light.</li>
		<li>Wash the cells twice with PBS and suspend them in 200 &#181;L of fixation buffer.</li>
		<li>Analyze the cells on a flow cytometer. 
		NOTE: The signal remains stable for several days if the samples are stored at 4 &#176;C and protected from light.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Open two dot-plots on a worksheet of the flow cytometer operating software. Set the FSC-A versus FSC-H in the first and the FSC-A versus SSA-A in the second. Open a histogram for the PE-Cy5 channel.</li>
		<li>In the FSC-A versus FSC-H plot, make a gate (R1) selecting singlet events (Figure 1A).</li>
		<li>Set the R1 population in the FSC-A versus SSA-A dot-plot and then make a new gate (R2) selecting target cells (Figure 1B). Set the R2 population in the PE-Cy5 intensity histogram to view the frequency distribution of the cells.</li>
		<li>Adjust the lower fluorescence intensity (FI) limit for the PE-Cy5 channel using the negative and isotype control (Figure 1C).</li>
		<li>Acquire 10,000 events within R2 from the sample with the higher concentration of the reference product. FI of this sample should be the highest expected (Figure 1C).</li>
		<li>Acquire the rest of the samples.</li>
		<li>For each sample, get the median fluorescence intensity (MFI) in the PE-Cy5 channel.</li>
		<li>For samples with the reference or biosimilar mAb, calculate the difference between sample MFI and that of the isotype control (&#916;MFI).</li>
	</ol>
	</li>
</ol>
2. Assessment of CDC
<ol>
	<li>Preparation of biological materials and reagents
	<ol>
		<li>Prepare cell culture medium and culture Daudi and Daudi GFP+ cells as described above (steps 1.1.1 - 1.1.2). 
		NOTE: Additionally, the CDC assay requires serum-free RPMI.</li>
		<li>Dilute normal human serum complement (NHSC) 1:2 with serum-free RPMI. Prepare 2.5 mL.</li>
		<li>Prepare 1 mL of heat-inactivated (30 min/56 &#176;C) NHSC diluted 1:2 with RPMI.</li>
		<li>Prepare sets of tests solutions for the reference and biosimilar mAbs in serum-free RPMI. Make ten dilutions (200 &#181;L each) from 1 to 0.025 &#181;g/mL.</li>
	</ol>
	</li>
	<li>CDC assay
	<ol>
		<li>Collect the Daudi and Daudi GFP+ cells from the cultures and quantify the cell viability (see steps 1.2.1 - 1.2.3).</li>
		<li>Prepare a cell suspension with 4 x 105 cells/mL in serum-free RPMI.</li>
		<li>Add 50 &#181;L of cell suspension to 50 &#181;L of each reference or biosimilar mAb test concentration in 96-well conical (V)-bottom microplates. Include replicates for each experimental condition.</li>
		<li>Prepare additional wells for the negative control (i.e., without mAb), basal death control (i.e., heat-inactivated NHSC in the presence of mAb), and staining positive control (i.e., cells exposed to 50 &#181;L of 70% EtOH).</li>
		<li>Incubate the cells for 20 - 30 min at 37 &#176;C in a 5% CO2 humidified atmosphere.</li>
		<li>Add 50 &#181;L of NHSC (diluted 1:2) to each well and incubate the opsonized cells for 2.5 h at 37 &#176;C in a 5% CO2 humidified atmosphere. Use heat-inactivated NHSC in the basal death control wells.</li>
		<li>Centrifuge at 400 x g for 5 min at 10 &#176;C. Discard the supernatant.</li>
		<li>Wash the cells by adding 150 &#181;L of PBS and centrifuging the cell suspension for 5 min at 400 x g and 10 &#176;C. Discard the supernatant.</li>
		<li>Stain the samples with 7-aminoactinomycin (7-AAD), as previously described7,8.</li>
		<li>Analyze the cells on a flow cytometer on the same day.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Open two dot-plots on a worksheet of the flow cytometer operating software. Set those plots as in steps 1.3.1 - 1.3.3 (Figure 2A-B). Create a third plot that is a dot-plot for GFP versus 7-AAD on the R2 population.</li>
		<li>Define the adequate FI limits using the Daudi cells, Daudi GFP+ cells, and death positive control (Figure 2C).</li>
		<li>For each sample, measure the percentage of 7-AAD+ target cells. Acquire at least 5,000 events from R2.</li>
		<li>Calculate the specific mAb-induced cytotoxicity by subtracting the percentage of 7-AAD+ in the basal death control from the percentage found in samples with different concentrations of mAbs (Figure 2D).</li>
	</ol>
	</li>
</ol>
3. Biosimilarity Analysis
<ol>
	<li>Enter the concentration and response values into a graphing software.</li>
	<li>Generate graphs and calculate non-linear regressions with the following considerations: i) use the log-transformation of the mAb concentration as &#34;X&#34;; ii) use the variable slope mathematical model (Y = minimum response + (maximal response - minimum response)/ 1+10^((LogEC50-X)*Hill slope)); and iii) constrain the bottom values to zero, since the basal response has been subtracted. 
	NOTE: Curves with a symmetrical sigmoidal shape are expected.</li>
	<li>Compare both non-linear fits with a global fit using an F-test (many graphing software programs include this feature). 
	NOTE: Such tests establish as the null hypothesis that the maximal response, logEC50, and the Hill slope are the same for the two datasets, which matches the biological question intended to be addressed.</li>
</ol>

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N. Salinas-Jazm&#237;n, E. Gonz&#225;lez-Gonz&#225;lez, and S. M. P&#233;rez-Tapia are employees of UDIBI, which performs biosimilarity studies for several pharmaceutical companies.

The patent expiration of a therapeutic mAb is promoting the development of biosimilars. Thus, there is a need for simple methods that can identify differences in clinically relevant activities of these products. CD20+ cultured cells were employed for the evaluation of two key functional characteristics of rituximab: target binding and CDC induction. The former activity requires the recognition of CD20 by the Fab region of the mAb, while the latter depends mainly on the interaction of the Fc region with its com...

Therapeutic antibodies are recombinant monoclonal antibodies (mAbs) developed for the treatment of different pathologies, including cancers, autoimmune and chronic diseases, neurologic disorders, and others1. Currently, the FDA has granted approval to more than 40 therapeutic mAbs, and more are expected to reach the market in the following years.
Rituximab is a high-affinity chimeric monoclonal IgG1 antibody approved for the treatment of CD20+ B-cell non-Hodgkin&#39;s lymphoma (NHL), CD20+ follicular NHL, chronic lymphocytic leukemia, and rheumatoid arthritis2,3. The recognition of CD20, which is overexpressed in B cells, by rituximab induces apoptosis; complement activation; and antibody-dependent cell mediated cytotoxicity (ADCC)3. The patents of this drug expired in Europe and in the U.S. in 2013 and 2016, respectively. Thus, pharmaceutical companies worldwide are developing rituximab biosimilars. As in any other drug for human consumption, biosimilars require approval from regulatory agencies. International guidelines indicate that for mAbs, biosimilarity should be demonstrated by comparing the physicochemical characteristics, pharmacokinetics, efficacy, and safety of the new and reference products4.
Accordingly, the methodologies used in such comparisons must assess the structural and functional characteristics of the mAbs, especially those with clinical relevance. To that end, in vitro assays show several advantages over in vivo experiments (reviewed in Chapman et al.)5: i) in vitro studies are more sensitive to differences between the proposed biosimilar and the reference product; ii) in vivo studies must be performed in relevant species, which for many mAbs are non-human primates; and iii) since the mechanism of action, the preclinical toxicology, and the clinical effects of the reference product are well known, in vivo studies with biosimilars may not provide additional useful information. Accordingly, the European Union&#39;s Guidance for biosimilars allows candidates to enter clinical trials based on robust in vitro data alone6.
Here, we present two fast, economic, and simple assays that evaluate the biological activity of rituximab using CD20+ cultured cells. These assays can be included as part of the comparability exercise for rituximab biosimilar candidates.

<table><tbody><tr><td>RPMI-1640 medium</td><td>ATCC</td><td>30-2001</td><td>Modify the culture depending on the cell line</td></tr><tr><td>Trypan Blue solution</td><td>Sigma</td><td>T8154</td><td>0.4%, liquid, sterile-filtered, suitable for cell culture</td></tr><tr><td>Daudi Burkitt&#039;s Lymphoma Cells</td><td>ATCC</td><td>CCL-213</td><td>You can modify the cell line depending on the antibody of interest</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GIBCO</td><td>16000-044</td><td>You can modify the source of serum depending of requirements of the cell line</td></tr><tr><td>Normal Human Serum Complement</td><td>Quidel</td><td>A113</td><td>It is therefore appropriate for use in biocompatibility experiments including drug development, biomaterials testing and other applications</td></tr><tr><td>7AA-D</td><td>BDPharmigen</td><td>559925</td><td>You can use broad range of color options, compatible with most instrument configurations for to analyze viability.</td></tr><tr><td>PECy5 Mouse Anti-human IgG</td><td>BDPharmigen</td><td>551497</td><td>Change fluorochrome depending on the filter and laser of your flow cytometer</td></tr><tr><td>Human IgG Isotype Control</td><td>ThermoFisher Scientific</td><td>07-7102</td><td>Change depending to mAb</td></tr><tr><td>BDCytofix</td><td>BDPharmigen</td><td>554655</td><td>Flow Cytometry Fixation Buffer (1 - 4% formaldehyde or paraformaldehyde )</td></tr><tr><td>PBS pH 7.4 10x (Phosphate buffer saline)</td><td>GIBCO</td><td>70011-044</td><td>Phosphatebuffer without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; [137 mM NaCl, 2.7 mM KCl, 8 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.46 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;] and endotoxin free.</td></tr><tr><td>Cell culture plates 96 well, V-bottom</td><td>Corning</td><td>29442-068</td><td>12 x 75 mm round bottom test tubes or 96-well V- or U-bottom microtiter plates</td></tr><tr><td>MabThera (Rituximab)</td><td>Roche</td><td>&amp;mdash;</td><td>Reference product</td></tr><tr><td>Rituximab</td><td>Indian</td><td>&amp;mdash;</td><td>Biosimilar product</td></tr><tr><td>15- or 50-mL conical centrifuge tubes</td><td>Corning</td><td>430290 or 430052</td><td>&amp;mdash;</td></tr><tr><td>Pipette Tips</td><td>Eppendorf</td><td>&amp;mdash;</td><td>Multiple volume configurations are necessary</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>&amp;mdash;</td><td>Adjustable-volume pipettes are necessary</td></tr><tr><td>Centrifuge 5430/ 5430R model</td><td>Eppendorf</td><td>&amp;mdash;</td><td>Refrigerated variable-speed centrifuge (4 to 25 &amp;deg;C) with speeds ranging from 10 to 30,130 &amp;times; g</td></tr><tr><td>Flow cytometer</td><td>BD Dickinson</td><td>&amp;mdash;</td><td>BD FACSAria III or other flow cytometer</td></tr><tr><td>Olympus optical and light microscope</td><td>Olympus</td><td>&amp;mdash;</td><td>To quantify and evaluate cell growth</td></tr><tr><td>Incubator</td><td>SANYO</td><td>&amp;mdash;</td><td>Incubatorfor temperature and CO&lt;sub&gt;2&lt;/sub&gt; control to culture cells</td></tr><tr><td>Biological Safety Cabinet</td><td>CHC BIOLUS</td><td>&amp;mdash;</td><td>Biological safety cabinet that is used to protect the researcher, product and environment.</td></tr></tbody></table>

in vitro methods for comparing target binding and cdc induction between therapeutic antibodies: applications in biosimilarity analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

Using the protocols described above, target binding and the CDC induction of reference rituximab were compared in parallel with those of a biosimilar rituximab produced and commercially available in Asia.
In Daudi cells, both mAbs bound CD20 in a concentration-dependent manner (Figure 1D). Non-linear regressions of binding data displayed an r2 of 0.978 and 0.848 for reference and biosimilar rituximab,...

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In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

in-vitro-methods-for-comparing-target-binding-cdc-induction-between

Research

JoVE Journal

Immunology and Infection

17.7K Views.  National Polytechnic Institute (IPN), University of Mexico (UNAM).  Dit protocol beschrijft de in vitro vergelijking van twee belangrijke functionele kenmerken van rituximab: target binding en complement-dependent cytotoxicity (CDC) inductie. De methoden werden gebruikt voor een vergelijking tussen referentie rituximab en een rituximab biosimilar. Deze analyses kunnen worden gebruikt bij biosimilar ontwikkeling of als kwaliteitscontrole in hun productie. 

Video: In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Biochemistry

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Cancer Research

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Medicine

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

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