Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

T cell-engaging bispecific antibodies (T-BsAbs) are engineered antibodies used to provide artificial specificity to polyclonal T cells by engaging T cells through one binding arm and a tumor antigen through another binding arm. This technology has been successfully applied to hematological cancers (CD19-targeting blinatumomab¹), and numerous T-BsAbs are in preclinical and clinical development for a variety of solid tumors as well². T-BsAbs engage polyclonal T cells in a major histocompatibility complex (MHC)-independent manner, and therefore even tumors that downregulate human leukocyte antigens (HLAs) are susceptible to this type of therapy^3,4. T-BsAbs have been developed in dozens of different formats, with differences in the valency and spatial arrangement of the T cell and tumor binding arms, interdomain distances, and the inclusion of an Fc domain, which affects the half-life and can induce effector functions if present⁵. Previous work in our lab has shown that these factors significantly affect the anti-tumor efficacy of T-BsAbs, with up to 1,000-fold differences in potency⁶. Through this work, we identified the IgG-[L]-scFv format as the ideal platform for T-BsAbs (see Representative Results section for more detail regarding T-BsAb formats), and have applied this platform to targets including GD2 (neuroblastoma), HER2 (breast cancer and osteosarcoma), GPA33 (colorectal cancer), STEAP1 (Ewing Sarcoma), CD19 (B cell malignancies), and CD33 (B cell malignancies)^{7,8,9,10,11,12,13}.

One of the major challenges to successfully implementing T-BsAb therapy in solid tumors is overcoming an immunosuppressive tumor microenvironment (TME) to drive T cell trafficking to tumors¹⁴. The factors affecting T-BsAb efficacy described above have a significant impact on the ability of T-BsAbs to effectively induce T cell homing to tumors, but this effect is difficult to evaluate in an in vivo system in real time. This manuscript provides a detailed description of the use of luciferase-transduced T cells in preclinical studies of T-BsAbs to evaluate T cell trafficking to various tissues in experimental immunocompromised mouse models during treatment. The overall goal of this method is to provide a means to evaluate T cell infiltration in tumors and other tissues, as well as real-time insight into T cell homing kinetics and persistence, without the need to sacrifice animals during treatment. For the increasing number of researchers focusing on cellular immunotherapies, the ability to track T cells in vivo in preclinical animal models is crucial. We aim to provide a thorough, detailed description of the method we have employed for tracking luciferase-transduced T cells to enable other researchers to easily replicate this technique.

The following procedures have been evaluated and approved by Memorial Sloan Kettering's Institutional Animal Care and Use Committee.

1. Transfection of 293T cells with luciferase and harvest of viral supernatant

1. Culture of 293T cells
   1. Prepare media by adding the following to a liter of DMEM each: 110 mL of heat-inactivated fetal bovine serum (FBS), 11 mL of penicillin-streptomycin.
   2. Thaw 5 x 10⁶ 293T cells and transfer them into a T175 flask with the media prepared as described above.
   3. Split the 293T cells 1:10 every 3 days for 6 days. Do not allow the cells to become more than 90% confluent.
2. 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.
   1. Transfer 1.5 mL of media to each of the two 50 mL tubes using a pipette. To one tube, add 10 µg of VSV-G plasmid, 20 µg of Gag/pol, and 20 µg of click beetle red TD tomato (CBR-TDR) luciferase plasmid and mix. To the other tube, add 100 µL of DNA in vitro 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.
   2. Transfer the media containing the DNA in vitro 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.
   3. 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 g for 5 min. Resuspend the cells in 1.5 mL of media.
   4. Add the media containing the DNA in vitro transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 °C for 30 min.
   5. Transfer to a T175 flask and add 18 mL of media. Incubate at 37 °C overnight.
   6. 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 °C overnight in an incubator.
3. Harvesting viral supernatant
   1. Remove the viral supernatant using a pipette put on ice. Centrifuge at 800 x g 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.
   2. Filter the viral supernatant using a 0.22 µm filter.
   3. Use the viral supernatant immediately. Keep it on ice or at 4 °C for up to 24 h, or freeze it at -80° C for long-term storage.

2. Expansion and transduction of activated human T cells with luciferase

1. Activation of human T cells in vitro
   1. 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.
   2. 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.
   3. 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.
   4. Plate 2.5 mL of media containing two million T cells, beads, and IL-2 in each well of a 24-well tissue culture plate. Culture at 37 °C in a humidified 5% CO₂ incubator.
   5. 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.
2. Preparation of retronectin plates
   NOTE: Retronectin is used to coat the plates used in the transduction as it enhances gene transduction¹⁵.
   1. Add 2 mL of 20 µg/mL retronectin in PBS to a well of an untreated 6-well plate. Incubate for 2 h at RT or 1 h at 37 °C. One retronectin-coated well is required for every two million T cells to be transduced.
   2. Aspirate the retronectin without scratching the bottom of the plate.
   3. Add 3 mL of 2% BSA in PBS (sterile filtered) per well in the 6-well plate. Incubate for 30 min at RT.
3. Spinoculation
   1. Aspirate the BSA without scratching the bottom of the plate.
   2. Wash the wells once with 3 mL of PBS per well. Continue or replace with fresh PBS and leave the plate covered at 4 °C overnight.
   3. Add 2 mL of CBR-TDR luciferase viral supernatant (collected in step 1.3) per well.
   4. Centrifuge at 1,240 x g for 90 min at 32 °C.
4. Transduction
   1. Count the T cells.
   2. Aspirate the viral supernatant without scratching the bottom of the plate.
   3. Plate two million T cells per well in 6 mL of Dulbecco's modified Eagle's medium (DMEM) containing 30 IU/mL human IL-2.
   4. Examine the T cells daily. The T cells are ready to be expanded when they are nearly confluent, usually after 2 days.
   5. 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.
   6. 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)¹⁶.

3. Engraftment of luciferase-transduced T cells in immunocompromised mice

1. Prepare and count the T cells for injection.
   1. 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.
   2. Centrifuge the T cells at 800 x g for 5 min. Resuspend in 2 mL of media and remove the beads using a magnet rack.
   3. 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 µL of media. Skip to step 3.2. For experiments using ex vivo armed T cells (EATs), skip to step 3.1.4.
   4. For experiments using ex vivo 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.
   5. Centrifuge the 1.5 mL tubes at 800 x g for 5 min. Remove the media carefully without disturbing the T cell pellet. Resuspend in no more than 50 µL 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 µg of antibody per 20 x 10⁶ T cells. Commercially available bispecific antibodies could also be used.
   6. Wash away excess antibodies by adding 1,450 µL of media to each tube. Centrifuge at 800 x g for 5 min, carefully aspirate the media, and resuspend at a concentration of 20 million armed T cells per 100 µL media.
2. Injection and engraftment into mice
   1. 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: Provide thermal support throughout the procedure.
   2. Using a 26 G needle, inject 20 million T cells in 100 µL of media retroorbitally per mouse.
   3. Administer 1,000 U recombinant IL-2 subcutaneously to support T cell survival in vivo.
   4. 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.
      ​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 µg of antibody per mouse per dose was administered. The antibodies were administered twice per week for 3-4 weeks.

4. In vivo 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.

1. Preparation of D-luciferin
   1. Dissolve 1 g of D-luciferin in 33.3 mL of sterile PBS (final concentration: 30 mg/mL).
   2. Aliquot the dissolved D-luciferin into 33 x 1.5 mL microcentrifuge tubes and keep the tubes at -20 °C.
   3. 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.
2. 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.
   1. 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.
   2. Once the mice are fully anesthetized, administer 100 µL 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.
3. Imaging luciferase-transduced T cells
   1. 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.
   2. Using the Acquisition Control Panel, select Luminescent, Photograph, and 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.
   3. Remove the mice from isoflurane, return to their cages, and observe until they are awake and ambulatory.
   4. Repeat the steps from step 4.2.1 until all groups have been imaged.

As described in step 4.3, mice can be oriented in different positions during imaging to evaluate the presence of T cells in different tissues. Supine positioning allows for the assessment of T cells in the lungs, which is common at early time points after injection. Lateral positioning with the subcutaneous xenograft facing up is used to best assess T cell trafficking to the tumor. Female C.Cg-Rag2^tm1Fwa Il2rg^tm1Sug/JicTac mice were used for all experiments described in this manuscript. Figure 1 shows images from an experiment in which mice bearing GD2-positive xenografts were treated with either luciferase-transduced T cells armed ex vivo with a GD2 BsAb or luciferase-transduced T cells and GD2 BsAb injected separately¹⁷. Figure 1A shows that armed T cells trafficked to the tumor faster than unarmed T cells (Figure 1B), leaving the lungs 2 days sooner. Figure 1C shows the quantification of this phenomenon. Figure 1E-G shows that it is possible to monitor T cell trafficking for at least 28 days post-injection of the luciferase-transduced T cells, allowing researchers to track T cell homing and persistence throughout the course of treatment, unlike other techniques such as immunohistochemistry (IHC) which allow only a snapshot of T cell infiltration.

Without the ability to assess T cell trafficking in vivo, researchers testing T-BsAbs are unable to determine whether or not T cells are infiltrating and persisting in tumors during treatment. If a treatment fails, without sacrificing animals and performing IHC, it can be difficult or impossible to know whether the failure was due to a lack of T cell trafficking, persistence, T cell exhaustion, or some other cause. Figure 2A-C shows an experiment in which mice bearing breast cancer patient-derived xenografts were treated with luciferase-transduced human T cells and HER2-targeted BsAbs bearing different mutations to silence Fc functions⁷. Figure 2C shows that treatment with the HER2 BsAb with an unsilenced Fc had no effect on tumor growth compared to a control BsAb, whereas treatment with HER2 BsAbs containing an N297A mutation alone or in combination with a K322A mutation were able to completely halt tumor growth. Figure 2A and Figure 1B show that in groups with the silencing mutations, T cells' luminescence signal reached a higher peak on day 3 post-injection and persisted at a higher level compared to control BsAb and unsilenced HER2 BsAb. Follow-up experiments showed that in mice treated with T cells and BsAbs with unsilenced Fcs, the T cells in perivascular regions of the lungs were surrounded by murine neutrophils and macrophages, suggesting an Fc-dependent sequestration of the BsAb-bound T cells which prevents migration to the tumor. The pro-tumorigenic effect of tumor-resident M2-polarized macrophages has been well described. Figure 3A,B shows that in mice bearing neuroblastoma patient-derived xenografts (PDXs), depletion of Ly6c-positive macrophages significantly increases T cell homing to tumors, resulting in decreased tumor growth and prolonged survival (Figure 3C)¹⁸. This effect is even more pronounced in osteosarcoma PDXs (Figure 3D).

In vivo T cell tracking also allows researchers to monitor how T cell trafficking kinetics are affected by other variables in antibody engineering, including structural configuration. As described in the introduction of this article, previous work in our lab underlined the importance of bivalency and cis orientation of the tumor- and T cell-binding arms for the in vivo efficacy of BsAbs. Figure 4 shows that among a panel of BsAbs targeting the tumor antigen STEAP1 (Figure 4A), the IgG-[L]-scFv format resulted in significantly more T cell infiltration on day 6 after the first dose of T cells, which persisted for the duration of treatment (Figure 4C)¹⁰. This phenomenon was not predicted by in vitro cytotoxicity assays (Figure 4B), emphasizing the importance of confirming in vitro results in in vivo models.

Figure 1: Armed T cells exhibit faster tumor homing kinetics than BsAb-directed unarmed T cells, rapidly bypassing lung sequestration. Luciferase-transduced T cells were expanded and armed with BsAb (Luc(+) GD2-EATs). Luc(+) GD2-EATs (10 µg of GD2-BsAb/2×10⁷ T cells) or Luc(+) unarmed T cells (2 x 10⁷ cells) with or without GD2-BsAb (10 µg) were administered intravenously into GD2-positive neuroblastoma PDX-bearing mice when the average tumor volume reached 100 mm³. (A) Bioluminescence images of GD2-EATs trafficking into tumors. (B) Bioluminescence images of GD2 BsAb-directed unarmed T cell trafficking into tumors over days. (C) Quantitation of T cell infiltration into tumors over time measured by bioluminescence (n = 5 mice/group) expressed as total flux or radiance (photons/s) per pixel integrated over the tumor contour (ROI). (D) Tumor growth curves of individual mice treated with GD2-EATs, GD2-BsAb plus unarmed T cells, or unarmed T cells. To test the in vivo persistence of target antigen-specific EATs, Luc(+) GD2-EATs (armed with 10 µg of GD2-BsAb/2×10⁷ T cells) or Luc(+) HER2-EATs (10 µg of HER2-BsAb/2×10⁷ T cells) were intravenously administered into osteosarcoma TEOS1C PDX-bearing mice. Two additional doses of non-luciferase transduced GD2-EATs or HER2-EATs were administered on day 7 and day 14. (E) Quantitation of bioluminescence of Luc(+) EATs in tumors post-treatment. (F) Tumor growth curves of individual mice treated with GD2-EATs, HER2-EATs, or unarmed T cells, or no treatment. (G) Bioluminescence images of Luc(+) GD2-EATs (upper) or Luc(+) HER2-EATs (lower) in tumors over time. Bioluminescence of Luc(+) EATs was detected over 28 days post-injection. Abbreviations: EATs = ex vivo armed T cells; ROI = region of interest. Error bars indicate the standard error of the mean. This figure has been modified from Park et al.¹⁷. Please click here to view a larger version of this figure.

Figure 2: T cell trafficking into HER2-positive human breast cancer PDX in DKO mice. (A) Representative bioluminescence images of T cell trafficking. (B) Quantitation of T cell trafficking into tumors over time by bioluminescence (n = 5 mice/group) expressed as total flux or radiance (photons/s) in each pixel integrated over the entire tumor contour (ROI). (C) HER2-positive breast cancer PDX (M37) growth (n = 5 mice/group) after treatment with Ctrl BsAb, HER2 BsAb, and its Fc mutants. The results shown are representative results from at least three independent experiments. The significant differences were calculated based on the area under the curve (AUC). Error bars indicate the standard error of the mean. This figure has been modified from Wang et al.⁷. Please click here to view a larger version of this figure.

Figure 3: The effects of monocyte depletion on BsAb directed T cell trafficking and in vivo anti-tumor response. (A) Luciferase transduced T cells (Luc(+) T cells) or luciferase transduced GD2-BsAb armed T cells (Luc(+) GD2-EATs) were administered with anti-Ly6C antibody to the mice bearing a neuroblastoma patient-derived xenograft (PDX). (B) Bioluminescence in the lesions of the tumor was monitored. The bioluminescence images on day 7 and quantification of the bioluminescence in the lesions of the tumor. (C) In vivo anti-tumor response by GD2-EATs with anti-Ly6C antibody was tested against neuroblastoma PDXs. (D) In vivo anti-tumor effect of GD2-EATs with anti-Ly6C antibody was tested against osteosarcoma PDXs, and long-term survival was analyzed. Error bars indicate the standard error of the mean. In vivo anti-tumor effect was compared by the AUC and survival curve analyses. Tumor-infiltrating lymphocytes were quantified using the AUC of bioluminescence. Differences between samples indicated in the figure were tested for statistical significance using a two-tailed Student's t-test for two sets of data and one-way ANOVA with Tukey's post hoc test for three or more sets of data. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. This figure has been modified from Park et al.¹⁸. Please click here to view a larger version of this figure.

Figure 4: Anti-tumor activities of anti-STEAP1 T cell-engaging bispecific antibody. (A) Schematic representation of the six structural formats of STEAP1 BsAb. (B) Antibody-dependent T cell-mediated cytotoxicity (ADTC) assay against EFT cell lines (TC32 ad TC71) using different formats of STEAP1 BsAb. Effector to target (ET) cell ratio was 10:1. (C) Bioluminescence imaging (BLI) of Luc(+) T cells armed with six different formats of STEAP1 BsAb on (a) day 6 and (b) day 18 post-treatment and quantitation of bioluminescence intensity in the lesions of the tumor. Luciferase transduced T cells (Luc(+) T cells) were armed with various formats of STEAP1 BsAb (10 µg of STEAP1 BsAb/2 x 10⁷ T cells) and administered with supplementary IL-2 (1,000 IU/dose) to the mice bearing EFT PDXs (ES15a), and the BLIfollowed. Abbreviations: EFT = Ewing sarcoma family of tumors; PDXs = patient-derived tumor xenografts; STEAP = six-transmembrane epithelial antigen of prostate. Error bars indicate standard error of the mean. Tumor-infiltrating lymphocytes were quantified by calculating the area under the curve of the bioluminescence. Differences between samples indicated in the figure were tested for statistical significance using one-way ANOVA with Tukey's post hoc test. **** p < 0.0001. This figure has been modified from Lin et al.¹⁰. Please click here to view a larger version of this figure.

While the T-BsAb blinatumomab has been approved for CD19-positive hematological malignancies, the successful implementation of T-BsAbs in solid tumors has proven much more difficult. Catumaxomab, a T-BsAb directed against epithelial cell adhesion molecule (EPCAM), was approved for the treatment of malignant ascites in ovarian cancer patients, but production of the drug was subsequently halted for commercial reasons¹⁹. No other T-BsAbs have been approved for solid tumors, underlining the challenges associated with this type of therapy. Toxicity due to cytokine release syndrome (CRS) is a common issue, although this can be managed with steroids and cytokine inhibitors. In addition to toxicity, a lack of efficacy is common in trials of T-BsAbs for solid tumors. Inducing T cell trafficking and persistence in the tumor has proven difficult, likely owing to various immunosuppressive factors in the TME¹⁴. However, even preclinically, it is often unclear why a T-BsAb that performs well in vitro fails to effectively treat tumors in vivo.

The method for tracking T cells in vivo in real time described in this article is useful to researchers for several reasons. Monitoring T cell homing provides mechanistic insight into why certain T-BsAbs succeed or fail by allowing researchers to determine when T cell trafficking has begun and for how long the T cells have persisted in the tumor during therapy. The method can be repeated at multiple time points, allowing researchers to plot the kinetics of T cell homing over the course of several weeks. (Representative results in Figure 1 show that T cells persist in the tumor for at least 28 days.) In vivo imaging of luciferase-transduced T cells also eliminates the need to sacrifice animals during treatment for histological assessment of T cell infiltration, reducing the overall cost and the number of animals needed per experiment. Because the imaging can be repeated as often as required, it also allows for a much easier and more thorough evaluation of T cell homing kinetics compared to histological analysis, which is essentially a snapshot of a single time point unless multiple animals are sacrificed at different time points.

The most critical step of this method is the successful transduction of T cells with the luciferase construct. Unsuccessful transduction can lead to T cell death or a lack of luciferase signal in vivo. If T cells are not expanding as expected after transduction, it may be possible to reduce the viral titer with which they are transduced in order to reduce potential toxicity from the transduction process. Another approach would be to wait another day between T cell expansion steps to allow for the T cells to become more confluent before expanding them into a larger cell culture vessel. Before injecting the T cells into the mice, it is a good idea to confirm transduction success using flow cytometry (to check for expression of the TD tomato reporter) or a bioluminescent plate reader. If the transduced T cells are still not visible with bioluminescent imaging, it may be possible that the number of T cells is too low for detection using this technique. Rabinovich et al. describe a similar method using enhanced firefly luciferase to detect as few as 10 murine T cells, which may be implemented in situations requiring this level of sensitivity²⁰.

In summary, in vivo tracking of luciferase-transduced T cells provides a valuable tool for researchers studying T-BsAbs in immunocompromised mouse models of cancer. In addition to the applications discussed above, this method of T cell tracking has been applied to chimeric antigen receptor (CAR) T cells and could ostensibly be applied to syngeneic mouse models utilizing adoptively transferred T cells as well²¹. We hope that this strategy will be helpful to researchers developing translational T-BsAbs and will lead to an increased success of these therapies in patients with solid tumors.