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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Transfection of 293T cells with luciferase and harvest of viral supernatant
<ol>
	<li>Culture of 293T cells
	<ol>
		<li>Prepare media by adding the following to a liter of Dulbecco&#39;s modified Eagle medium (DMEM) each: 110 mL of heat-inactivated fetal bovine serum (FBS), 11 mL of penicillin-streptomycin.</li>
		<li>Thaw 5 x 106&#160;293T cells and transfer them into a T175 flask with the media prepared as described above.</li>
		<li>Split the 293T cells 1:10 every 3 days for 6 days. Do not allow the cells to become more than 90% confluent.</li>
	</ol>
	</li>
	<li>Transfection of 293T cells 
	NOTE: The following quantities of reagents are calculated for transfecting one T175 flask of 293T cells. Adjust accordingly if more flasks are to be transduced.
	<ol>
		<li>Transfer 1.5 mL of media to each of the two 50 mL tubes using a pipette. To one tube, add 10 &#181;g of VSV-G plasmid, 20 &#181;g of Gag/pol, and 20 &#181;g of click beetle red TD tomato (CBR-TDR) luciferase plasmid and mix. To the other tube, add 100 &#181;L of DNA&#160;in vitro&#160;transfection reagent and mix. Incubate both tubes at room temperature (RT) for 5 min. 
		NOTE: All plasmids described in this manuscript were provided by Dr. Vladimir Ponomarev.</li>
		<li>Transfer the media containing the DNA&#160;in vitro&#160;transfection reagent dropwise to the tube containing the DNA plasmids (over approximately 30 s) and mix gently with a pipette. Incubate at RT for 20 min.</li>
		<li>During this 20 min incubation, detach the 293T cells by adding 5-10 mL of 0.05% trypsin. Once the cells have detached, add 10 mL of media and centrifuge at 800 x&#160;g&#160;for 5 min. Resuspend the cells in 1.5 mL of media.</li>
		<li>Add the media containing the DNA&#160;in vitro&#160;transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 &#176;C for 30 min.</li>
		<li>Transfer to a T175 flask and add 18 mL of media. Incubate at 37 &#176;C overnight.</li>
		<li>The next day, carefully aspirate the media with a glass pipette without detaching the cells. Replace with 18 mL of fresh media. Incubate at 37 &#176;C overnight in an incubator.</li>
	</ol>
	</li>
	<li>Harvesting viral supernatant
	<ol>
		<li>Remove the viral supernatant using a pipette put on ice. Centrifuge at 800 x&#160;g&#160;for 5 min to pellet the cells and debris. 
		NOTE: If the cell pellet is large, the cells were likely disrupted from the flask when the supernatant was removed. These cells can be plated again in a new flask with fresh media.</li>
		<li>Filter the viral supernatant using a 0.22 &#181;m filter.</li>
		<li>Use the viral supernatant immediately. Keep it on ice or at 4 &#176;C for up to 24 h, or freeze it at -80&#176; C for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Expansion and transduction of activated human T cells with luciferase
<ol>
	<li>Activation of human T cells&#160;in vitro
	<ol>
		<li>Wash the beads by adding 10 mL of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA) to a sterile 15 mL tube, adding beads (one bead per two T cells), placing in a magnet rack for 2 min, and suctioning out the PBS.</li>
		<li>Prepare media as described in step 1.1.1, then add IL-2 to a final concentration of 30 IU/mL and add the washed beads. Make 2.5 mL of media for every two million T cells to be activated.</li>
		<li>Count the T cells (freshly purified or previously purified, frozen, thawed, and washed) using a hemocytometer and trypan blue stain. Add T cells to the media prepared in step 2.1.2 and gently invert the tube to mix. The T cells will expand at least 20x depending on the source and general health of the cells. Determine the number of T cells to activate by calculating the number needed to treat the mice and dividing by 20. Here, 20 x 106 T cells per mouse were used based on preliminary experiments.</li>
		<li>Plate 2.5 mL of media containing two million T cells, beads, and (interleukin 2) IL-2 in each well of a 24-well tissue culture plate. Culture at 37 &#176;C in a humidified 5% CO2&#160;incubator.</li>
		<li>Examine the T cells under a 20x microscope every day for the next 3 days to determine when to transduce with luciferase. The cells are ready when they clump together and deplete the media, turning it from red/pink to light orange.</li>
	</ol>
	</li>
	<li>Preparation of retronectin plates 
	NOTE: Retronectin is used to coat the plates used in the transduction as it enhances gene transduction.
	<ol>
		<li>Add 2 mL of 20 &#181;g/mL retronectin in PBS to a well of an untreated 6-well plate. Incubate for 2 h at RT or 1 h at 37 &#176;C. One retronectin-coated well is required for every two million T cells to be transduced.</li>
		<li>Aspirate the retronectin without scratching the bottom of the plate.</li>
		<li>Add 3 mL of 2% BSA in PBS (sterile filtered) per well in the 6-well plate. Incubate for 30 min at RT.</li>
	</ol>
	</li>
	<li>Spinoculation
	<ol>
		<li>Aspirate the BSA without scratching the bottom of the plate.</li>
		<li>Wash the wells once with 3 mL of PBS per well. Continue or replace with fresh PBS and leave the plate covered at 4 &#176;C overnight.</li>
		<li>Add 2 mL of CBR-TDR luciferase viral supernatant (collected in step 1.3) per well.</li>
		<li>Centrifuge at 1,240 x&#160;g&#160;for 90 min at 32 &#176;C.</li>
	</ol>
	</li>
	<li>Transduction
	<ol>
		<li>Count the T cells.</li>
		<li>Aspirate the viral supernatant without scratching the bottom of the plate.</li>
		<li>Plate two million T cells per well in 6 mL of DMEM containing 30 IU/mL human IL-2.</li>
		<li>Examine the T cells daily. The T cells are ready to be expanded when they are nearly confluent, usually after 2 days.</li>
		<li>When the cells are nearly confluent, gently wash them off the bottom of the plate using a pipette and transfer each well to a separate T75 flask. Add 9 mL of media for a total of 15 mL of media per flask.</li>
		<li>The next day, add an additional 15 mL of media. The cells should be ready to inject into mice the day after this addition of media. Confirm successful transduction by performing flow cytometry (the luciferase construct contains a TD tomato fluorophore that is visible in the PE channel).</li>
	</ol>
	</li>
</ol>
3. Engraftment of luciferase-transduced T cells in immunocompromised mice
<ol>
	<li>Prepare and count the T cells for injection.
	<ol>
		<li>Remove the T cells from the flasks and count. The cells are ready to inject when they have expanded 20x-30x, usually by day 7 post-activation.</li>
		<li>Centrifuge the T cells at 800 x&#160;g&#160;for 5 min. Resuspend in 2 mL of media and remove the beads using a magnet rack.</li>
		<li>For experiments where T cells will be injected separately from bispecific antibodies, count the T cells and resuspend so that the final concentration is 20 million T cells per 100 &#181;L of media. Skip to step 3.2. For experiments using&#160;ex vivo&#160;armed T cells (EATs), skip to step 3.1.4.</li>
		<li>For experiments using&#160;ex vivo&#160;armed T cells, count the T cells and separate them into individual 1.5 mL tubes for each group, with 20 million cells per mouse. For example, if there are three groups of five mice each, prepare three 1.5 mL tubes with 100 million T cells each.</li>
		<li>Centrifuge the 1.5 mL tubes at 800 x&#160;g&#160;for 5 min. Remove the media carefully without disturbing the T cell pellet. Resuspend in no more than 50 &#181;L of media containing the bispecific antibodies. Incubate at RT for 30 min. 
		NOTE: For the purposes of experiments conducted in this study, proprietary IgG-[L]-scFv T cell-engaging bispecific antibodies generated in the lab were used. For T cell arming, use 5 &#181;g of antibody per 20 x 106&#160;T cells. Commercially available bispecific antibodies could also be used.</li>
		<li>Wash away excess antibodies by adding 1,450 &#181;L of media to each tube. Centrifuge at 800 x&#160;g&#160;for 5 min, carefully aspirate the media, and resuspend at a concentration of 20 million armed T cells per 100 &#181;L media.</li>
	</ol>
	</li>
	<li>Injection and engraftment into mice
	<ol>
		<li>Anesthetize the mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared. 
		NOTE:&#160;Provide thermal support throughout the procedure.</li>
		<li>Using a 26 G needle, inject 20 million T cells in 100 &#181;L of media retroorbitally per mouse.</li>
		<li>Administer 1,000 U recombinant IL-2 subcutaneously to support T cell survival&#160;in vivo.</li>
		<li>Administer the bispecific antibodies retroorbitally (into the eye not used for T cell injection) or intraperitoneally. Allow the mice to recover from anesthesia and return to their cages. 
		&#8203;NOTE: Again, here, proprietary antibodies that were generated in the lab were used, but commercially available antibodies could be used instead. In the experiments here, 0.3-10 &#181;g of antibody per mouse per dose was administered. The antibodies were administered twice per week for 3-4 weeks.</li>
	</ol>
	</li>
</ol>
4.&#160;In vivo&#160;imaging of mice engrafted with luciferase-transduced T cells
NOTE: This step is to be performed on the day of imaging, which is not necessarily the same day that the T cells and/or antibodies are administered to the mice. Typically, we perform imaging 24 h after administration of the luciferase-transduced T cells.
<ol>
	<li>Preparation of D-luciferin
	<ol>
		<li>Dissolve 1 g of D-luciferin in 33.3 mL of sterile PBS (final concentration: 30 mg/mL).</li>
		<li>Aliquot the dissolved D-luciferin into 33 x 1.5 mL microcentrifuge tubes and keep the tubes at -20 &#176;C.</li>
		<li>On the day of imaging, thaw enough aliquots of 30 mg/mL D-luciferin for the number of animals to be imaged. One tube is enough for 10 animals. 
		NOTE: Refreeze the remaining D-luciferin after use. D-luciferin is stable for at least five freeze/thaw cycles.</li>
	</ol>
	</li>
	<li>Administration of D-luciferin to mice 
	NOTE: Before administering D-luciferin to the mice, open the imaging software and initialize the system. The camera may take several minutes to cool, and this should be done before the mice are injected with D-luciferin to ensure that imaging can take place 5 min after administration of the substrate.
	<ol>
		<li>Anesthetize up to five mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared.</li>
		<li>Once the mice are fully anesthetized, administer 100 &#181;L of 30 mg/mL D-luciferin per mouse (3 mg per mouse) by retroorbital injection with a 26 G needle. Image this group of mice before administering D-luciferin to the next group. Place the next group of mice in the isoflurane chamber to be anesthetized while the previous group is being imaged.</li>
	</ol>
	</li>
	<li>Imaging luciferase-transduced T cells
	<ol>
		<li>Move the anesthetized mice to the light-tight chamber of the imager and continue administering 3% isoflurane at 0.5 L/min. Place the mice on their sides such that the flank bearing the xenograft is facing up toward the camera. Take another set of images with the mice in a supine position to evaluate T cell presence in the lungs.</li>
		<li>Using the&#160;Acquisition Control Panel, select&#160;Luminescent,&#160;Photograph, and&#160;Overlay. Set the exposure time to auto, the binning to medium, and F/Stop to 1. Acquire images. After the first image, set the exposure time to match that which was automatically calculated for the first image so that subsequent images may be compared directly. It may be useful to capture images with multiple exposure times to determine the optimal exposure time for achieving the brightest signal without pixel saturation.</li>
		<li>Remove the mice from isoflurane, return to their cages, and observe until they are awake and ambulatory.</li>
		<li>Repeat the steps from step 4.2.1 until all groups have been imaged.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3/CD28 beads</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11161D</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA in vitro transfection reagent (polyjet)</td>
			<td>SignaGen Laboratories</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (ThermoFisher)</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gag/pol plasmid</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP plasmid</td>
			<td>Addgene</td>
			<td>11150-DNA.cg</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G plasmid</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo technique to track bispecific antibody-induced t cell trafficking to tumors

Source: Espinosa-Cotton, M., et al. <a href="https://app.jove.com/v/64390">Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells.</a> J. Vis. Exp. (2023).
This video demonstrates a method for tracking bispecific antibody-induced T cell trafficking to tumors in a mouse model. Luciferase-transduced effector T cells are injected into a mouse with a subcutaneous tumor cell xenograft. Bispecific antibodies are then introduced, binding to T cells and the tumor cells. The localization of the T cells is tracked using bioluminescent imaging, with the magnitude of the signal indicating the extent of T cell infiltration into the tumor.

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take an anesthetized, immunocompromised mouse bearing a subcutaneous tumor xenograft that overexpresses a tumor-associated antigen or TAA.
Retroorbitally inject effector T cells expressing the luciferase enzyme.
Subcutaneously administer interleukin 2, a cytokine supporting effector T cell survival.
Retroorbitally inject bispecific antibodies engineered to contain binding sites for T and tumor cells. Allow the mouse to recover.
The antibodies and T cells reach the tumor via circulation.
The antibodies bind to CD3 on the T cells and TAA on the tumor cells, inducing the effector T cells to release toxins that initiate apoptosis in the tumor cells and release chemokines that recruit more T cells to the tumor.
Anesthetize the mouse, retroorbitally inject a luciferase substrate, and proceed to in vivo imaging.
Luciferase inside the T cells oxidizes the substrate, emitting bioluminescence.
Observe the bioluminescence over time. The magnitude of the signal correlates with the extent of T cell infiltration within the tumor.

an-vivo-technique-to-track-bispecific-antibody-induced-t-cell

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

83 조회수. 출처: Espinosa-Cotton, M., et al. 루시페라아제 형질도입 인간 T 세포를 사용한 이중특이성 항체 유도 T 세포 트래픽 추적. J. Vis. 특급 (2023). 이 비디오는 마우스 모델에서 종양으로의 이중특이성 항체 유도 T 세포 이동을 추적하는 방법을 보여줍니다. 루시페라아제(Luciferase)에 의해 형질도입된 effector T 세포를 피하 종양 세포 이종이식편을 통해 마우스에 주입합니다. 그런 다음 이중?...

비디오: 종양에 대한 이중특이성 항체 유도 T 세포 이동을 추적하는 In Vivo 기술 - 개념

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

GPC3 대상으로 Bispecific 항 체, GPC3-S-팹, 강력한 세포 독성을

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

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JoVE Journal

암 연구학

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

Alpacas (라마 pacos) 단백질 항 원 및 항 원 특정 단일 도메인 항 체의 생산의 면역

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

생화학

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

키메라 항원 수용체 T 세포의 동적 이미징 [18F]테트라플루오로보레이트 양전자 방출 단층 촬영/컴퓨터 단층 촬영

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors

내레이션 대본

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors