Third-Generation Human Epidermal Growth Factor Receptor 2 Chimeric Antigen Receptor Expression on Human T Cells Improves with Two-Signal Activation

Abstract

Patient derived T cells activated ex vivo with CD3/CD28 beads show superior expansion. Therefore, CD3/CD28 beads have huge potential to be used in the clinic for immunotherapy applications. Two protocols were devised to evaluate if the expression of third-generation human epidermal growth factor receptor 2 chimeric antigen receptor (CAR) can be improved on human T cells activated with CD3/CD28 beads. In protocol 1, unconcentrated human epidermal growth factor receptor 2 CAR retroviral supernatants were used, and in protocol 2, concentrated virus was used. The results demonstrate that compared to unconcentrated viral supernatants, transduction with the concentrated virus improved the infection rate of bead activated CD4 T cells from ∼40% to ∼70%, and the fluorescent intensity values improved from ∼12,000 to ∼28,000 mean fluorescence intensity units. These results demonstrate the utility of these protocols for CAR immunotherapies.

Introduction

T cells are biologically programmed to target tumors. The antitumor role of T cells is achieved through direct help of type I cytokines such as interleukin (IL)-2, which is synthesized and secreted by CD4 and CD8 T cells in response to antigen presentation through major histocompatibility complex (MHC) molecules. IL-2 subsequently signals to CD8+ and natural killer (NK) cells, promoting their survival and cytolytic activity against cancer cells. Thus, the natural route for T cells to receive signals about tumor presence is MHC restricted. The MHC restriction may be one factor limiting aggressive antitumor responses. However, MHC restrictions can be overcome successfully when the knowledge of antibody specificity is combined with cytotoxic T cells or NK cells properties to kill cancer cells. An antibody specific for tumor antigens such as human epidermal growth factor receptor 2 (HER2), if engineered to express on T cells, can allow T cells to access tumors without the need for MHC presentation. Such engineered T cells are referred to as chimeric antigen receptor (CAR) T cells.^1
–3

T cells modified genetically to express CARs are becoming an attractive immunotherapy tool that has shown great success in eradicating B-cell malignancies.^4

–7 However, the success story of CD19-CAR has rarely been replicated in CARs developed to treat solid tumors, as recorded in at least 13 clinical trials to data.⁸ For example, CAR T cells tested in clinical trials against ovarian cancer, metastatic renal carcinoma, and neuroblastoma have shown very limited persistence in vivo and partial clinical responses.^9

–13 Despite their limited success against solid tumors, CAR T-cell therapy remains very promising, and intense efforts are being invested to improve their efficacy. Various ways of generating CARs have been created to improve their efficacy.^14

–17

Generation of CARs

A prototypical first-generation CAR is composed of an extracellular antigen-binding domain and intracellular signaling domain. The extracellular domain is composed of a single-chain variable fragment (scFv) that binds to tumor-associated antigens. The intracellular signaling domain is composed of CD3ζ signaling domain. Extracellular and intracellular domains are linked by a hinge (spacer) and a transmembrane domain.¹⁸ First-generation CAR-modified T cells have shown limited antitumor cytotoxicity in vivo, and have been shown to undergo activation-induced cell death. Second-generation CARs apart from containing antigen binding scFv domain (signal-1) also incorporate co-stimulatory CD28 or 4I-BB (signal-2).¹⁸ The incorporation of co-stimulatory signal-2 has rendered modified T cells resistant to activation-induced cell death. Such T cells secrete type I cytokines and provide a prolonged antitumor response in vivo.^15,19,20 Third-generation CARs incorporate two co-stimulatory molecules.²¹ An example of a third-generation CAR is the human epidermal growth factor receptor 2 (HER2-CAR),²² which was developed specifically to target carcinoma.

HER2/neu is a tyrosine kinase receptor encoded by the ERBB2 gene. It is a member of the epidermal growth factor receptor family that upon ligand interaction leads to the heterodimerization of receptors and intracellular kinase signaling that leads to cell growth, differentiation, and survival.²³ HER2 overexpression can lead to aberrant intracellular kinase signaling cascade without ligand interaction. HER2 overexpression is associated with several cancers such as ovarian, stomach, lung, uterus, and salivary duct carcinoma.^24,25 Moreover, HER2 overexpression is reported in 18–20% of all breast cancer cases and is associated with a poor prognosis.^25,26 Various ways of generating HER2-CARs have been specifically developed to target ERBB2-positive breast cancers.^27,28 In particular, third-generation HER-CARs have shown promising results in both in vitro and in vivo animal models.^27,28 For example, Herceptin-based humanized HER2 third-generation CAR has shown superior effector functions, including effector cytokine production, compared to second-generation Herceptin CAR.²⁷ Nevertheless, solid preclinical data would be extremely useful for the promise of better clinical outcome in patients. However, poor ex vivo expansion of patient-derived T cells can impose a bottleneck in obtaining solid preclinical data. A powerful new technology that develops clinical grade CD3/CD28 beads has shown better ex vivo activation and expansion of human T cells. Therefore, this new technology has the potential to be applied in clinical applications, especially for ex vivo expansion of patient-derived T cells and CAR immunotherapy.^29,30

Several biotech companies have begun to manufacture clinical grade CD3/CD28 beads for ex vivo expansion of T cells. These include CTS CD3/CD28 Dynabeads, the Miltenyi MACS GMP ExpAct Treg beads, and Miltenyi MACS GMP TransAct CD3/CD28 beads. The CD3/CD28 Dynabeads produced by Thermo Fisher Scientific can produce 100- to 1,000-fold expansion of T cells in a relatively short time frame. Furthermore, studies have already established the utility of this technology while working with large sample sizes.³¹ Therefore, protocols was designed specifically to evaluate if the expression of third-generation HER-CARs on human T cells can be improved with bead activation. The third-generation HER2-CAR that was tested in these protocols is lentiviral based and has been genetically modified to include mCherry Red fluorescent protein to help sort CAR-positive cells through flow cytometry, besides allowing a straightforward estimation of the percent modification of target cells (Fig. 1).

Figure 1.

Design of third-generation human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR). (A) The design and structure of third-generation lentivirus based HER2-CAR. Each LTRs have their own promoters whereas SFFV drives the expression of HER2-CAR. mCherry fluorescent gene is fused with CD3ζ signaling sequence. (B) The protein structure of HER-CAR.

Human T cells were subjected to modified viral transduction protocols combined with two-signal (signal 1 and 2) activation delivering Dynabeads that induced a greater proliferation rate compared to conventional OKT3 activation. The experiments achieved an effective expansion of CD4 T cells using CD3/CD28 beads, which is consistent with the results obtained by Li et al.³² An additional advantage of bead activation of the T cells was an improvement in CAR expression. When bead-activated T cells were transduced with viral supernatants that were subjected to a concentration protocol, overall, 70% CAR-modified T cells and mean fluorescence intensity (MFI) values in the range of 28,000 units were observed (Fig. 2). These values represent significant improvement in CAR expression compared to the traditional OKT3 activation method, indicating that ex vivo expansion of primary T cells with beads can be much more efficient and permissive for CAR expression. Whether the T cells using clinical grade bead activation and expressing a higher percentage of third-generation CARs can translate into a better clinical response remains to be seen. It is not inconceivable that improved CAR expression on T cells followed by better ex vivo expansion can help to scale up the production of CAR T cells for clinical use. However, high expressing CAR-modified T cells designed to target tumor associated antigens on solid tumors may be associated with the pitfall of off-target toxicities. Therefore, a carefully controlled and extensive preclinical evaluation of such CARs must be performed before moving to actual clinical use.

Figure 2.

HER-CAR expression on T cells activated with CD3/CD28 Dynabeads. (A) Morphology of T cells during CD3/CD28 bead activation. Images were captured with a Carl Zeiss Axioskop microscope (Axioskop 40; Carl Zeiss, Inc.). (B and C) demonstrate percent CAR modification of CD4 T cells. (B) Unconcentrated virus (protocol 1). (C) Concentrated virus (protocol 2). (D) Mean fluorescence intensity values.

Experimental Procedure

To test the protocols, enough HER-CAR lentiviral particles we produced and collected. The HER2-CAR vector was a kind gift from Dr. Wilson Wong at Boston University. HER2-CAR incorporates humanized anti-HER2 scFv. Humanized anti-HER2-scFv is composed of the variable region of an immunoglobulin heavy chain and the variable region of the light chain linked through a polypeptide linker. The hinge is composed of the human CD8α chain, which is further fused with a CD28 co-stimulatory molecule, part of which spans the transmembrane and cytoplasm. CD28 in turn is fused with a second co-stimulatory molecule, 4-1BB and CD3ζ signaling endodomains. The CAR expression is under the control of SFFV promoter. HER2-CAR is labeled with mCherry at the C-terminus of CD3ζ to help sort CAR-modified cells and to aid in various downstream applications (Fig. 1).

HER-CAR lentivirus production

The following protocol was used to produce HER-CAR lentiviral particles.

All media required for the lentiviral production were prepared prior to the procedure.

Day 0

Low-passage HEK293FT cells were seeded in a sterile T175 flask containing 20 mL of 10PSGN medium.

Day 1

At 60–80% confluency, HEK293FT cells were transfected with the combination of plasmids, pCMVR8.74, VSVG, pAdv, and pHR-SFFV HER2-CAR. Provided below is the transfection protocol for one reaction:

Polyethylenimine (PEI) transfection reagent and 0.15 M NaCl were briefly vortexed and warmed to room temperature before use.

PEI master mix was prepared by adding 350 μL of PEI to 900 μL of 0.15 M NaCl.

DNA plasmid mix was prepared by transferring 22 μg of viral plasmids to the tube containing 1.25 mL of 0.15M NaCl. Plasmids were added in the following ratio: 15 (pCMVR8.74):5 (VSV-G):2 (pAdv) and 22 μg of pHR-SFFV HER2-CAR plasmid. Tube was vortexed briefly.

The PEI mix (1.25 mL) prepared in (2) was added to the plasmid mix prepared in (3). The total volume in the tube was maintained around 2.50 mL.

The plasmid–PEI mix was incubated for 10 min at room temperature.

During the 10 min incubation time, the culture media from the HEK293FT flask was removed, and 7.5 mL of fresh, pre-warmed 10PSGN media was added.

The plasmid–PEI mix was gently added to the flask containing HEK293FT, and the flask was swirled gently to allow the plasmid mix to spread evenly over the cells.

The cells were placed in an incubator at 37°C for 24 h.

Day 2

Twenty-four hours post transfection, the medium of the cells was replaced with 20 mL of fresh, pre-warmed UPSGBN media, and the flask was transferred back to the incubator.

Day 3

The viral supernatant was collected from the flask and transferred to 50 mL falcon tubes placed on ice. Fresh, pre-warmed UPSGBN media (20 mL) can be added to the cell culture flask to collect more virus.

The collected viral supernatants were centrifuged to pellet the cell debris. The viral supernatants were subsequently filter sterilized over Beckman ultracentrifuge tubes using 0.45 μm sterile filters. Filter sterilized viral supernatant was stored at 4°C for immediate use. For long-term storage, small aliquots were made and stored in −80°C.

Note: Lab personnel handling retroviral production and performing infection of primary human T cells were thoroughly trained in BSL2 lab practices. All retroviral transduction procedures were carried out in BSL2 equipped lab. National Institutes of Health guidelines were followed for recombinant and synthetic nucleic acid molecules, and institutional policies relating to this project.

Isolation of primary human T cells and bead activation

Heparinized peripheral blood from healthy donors was obtained from Boston Children's Hospital blood bank. CD4 T cells were enriched from peripheral blood mononuclear cells (PBMCs) using RosetteSep enrichment mixtures by negative selection (cat. 15063; STEMCELL Technologies, Cambridge, MA). The enriched CD4+ T cells were activated with CD3/CD28 Dynabeads for 48 h (Fig. 2A). The cell-to-bead ratio was maintained at 1:3. The activation was followed by the viral transduction protocols.

Note: Blood samples were regarded as potential biohazard and handled with precautions and using all the protective equipment as recommended by the institutional guidelines. The needles and disposable plastic wares in contact with the blood specimens were discarded into biohazard labeled containers.

Protocol 1

Non-tissue culture six-well plates were coated with retronectin for 2 h at room temperature, after which the plates were washed with phosphate-buffered saline (PBS).

Before starting the actual transductions, the CD3/CD28 beads attached to the T cells were removed using a magnet. Subsequently, cells were counted and spun in a centrifuge at 450 g for 5 min, and an estimated 250 × 10³ cells were used for the viral transduction, as described in the steps below.

T cells prepared in step (2) above were suspended in 500 μL of HER2-CAR virus supernatant diluted in 1.5 mL (1:4 dilution) of T-cell growth medium supplemented with 100 IU/mL of IL-2 and protamine sulfate (5 μg/mL). This 2 mL solution containing diluted virus and bead-activated T cells was transferred to the retronectin-coated plates. Plates were sealed with Parafilm, balanced, and spun in a centrifuge at 2,000 g for 2 h at room temperature.

After centrifugation, seals were removed, and plates were transferred back to an incubator for a 3 h incubation at 37°C. After the end of the incubation period, plates were removed from the incubator, and the entire content in the wells was transferred to a falcon tube for 5 min centrifugation at room temperature. After centrifugation, the supernatant was carefully aspirated and discarded. The cell pellet was suspended in a fresh diluted virus, as described in step (3) and transferred back to the original retronectin-coated plates. Plates were sealed and subjected to second round of centrifugation at 2,000 g for 2 h at room temperature.

After the second centrifugation, the plate seal was removed, and the plates were placed back into an incubator for another 3 h at 37°C.

After the 3 h incubation period, 2 mL of fresh medium containing 100 IU/mL of IL-2 was added to the plate and placed back in an incubator at 37°C without applying any additional centrifugation. After 48 h, cells were subjected to flow cytometry analysis to determine the HER2-CAR expression through mCherry fluorophore detection.

The FACS data obtained with protocol 1 revealed 40% HER2-CAR-modified T cells compared to the 1.6% observed in the unactivated T cells subjected to the same transduction protocol (Fig. 2B). The MFI values were in the range of ∼12,000, which is a significant improvement over the unactivated controls (∼700; Fig. 2D) and compared to the OKT3 activation (data not shown). The lab has been routinely using OKT3 activation for viral transductions that usually results in 10–20% CAR-modified T cells (not shown).

Protocol 2

In the protocol 2, a viral concentration step was performed before the actual transduction of CD3/CD28 bead-activated CD4 T cells. The viral concentration protocol is described below.

Virus concentration

The procedure describes the concentration of 50 mL of batch viral supernatant.

The freshly collected HER2-CAR viral supernatant was filter sterilized over Beckman ultracentrifuge tubes using 0.45 μm sterile filters.

Filter sterilized 20% sucrose (2 mL) was gently added to a tube containing 50 mL of filter sterilized viral supernatant.

Tubes were subjected to ultracentrifugation using a sterilized SW28 rotor at 64,047 g for 2 h at 4°C.

After centrifugation, tubes were placed on ice. Supernatants were carefully aspirated off without disturbing the viral pellet.

Growth medium (300–500 μL, depending upon the size of the pellet) was added carefully to the pellet. The pellet in the growth medium was left undisturbed for 2 h at 4°C, after which the viral pellet was gently suspended in the growth medium using the pipette and avoiding the bubbles. The concentrated viral aliquots were transferred into sterilized Eppendorf tubes.

Viral aliquots were stored at 4°C for immediate use. For long-term storage, aliquots were transferred to −80°C.

Virus transduction

A non-tissue culture six-well plate was treated with retronectin at a concentration of 32 μg/mL. Coated plates was incubated for 2 h at room temperature.

After the 2 h incubation, retronectin solution was aspirated, and the plate wells were blocked for 30 min with 2 mL of blocking solution (2% bovine serum albumin dissolved in 1 × PBS) at the room temperature.

Plate wells were washed with 1 × PBS.

Concentrated HER-CAR viral aliquots (3.5 mL) were transferred to retronectin-coated wells.

The plate was sealed and centrifuged at 1,200 g for 90 min. During the spin, the CD3/CD28 bead-activated T cells maintained at a 1:3 cell-to-bead ratio and activated for 48 h were removed from the incubator. Cells were transferred into a 15 mL falcon tube, and the beads were removed using a magnet. The cells were counted, and approximately 250,000 cells/mL was resuspended in T-cell growth media supplemented with 100 IU/mL of IL-2.

After centrifugation, viral supernatant was aspirated from the retronectin-coated wells.

The bead-activated T cells at a concentration of ∼250,000 cells/mL were transferred onto the retronectin-coated plate and subjected to centrifugation at 1,200 g for 1 h applying a reduced centrifuge breaking speed (deceleration = 3).

After centrifugation, the plates were transferred to an incubator (maintained at 37°C and 5% CO₂ level).

Cells were analyzed for CAR expression 48 h post transduction.

The FACS data obtained with protocol 2 demonstrated ∼70% HER2-CAR-modified T cells (Fig. 2C). In comparison, only ∼40% CAR-modified T cells were achieved with protocol 1 (Fig. 2B). Likewise, the MFI values obtained with protocol 2 were in the range of ∼28,000 compared to only ∼12,000 MFI units obtained with protocol 1 (Fig. 2D). Overall, with protocol 2, around a twofold improvement was achieved in the percentage of HER2-CAR-modified T cells and in the MFI values.

Reagents and Consumables for Lentivirus Production

For lentiviral production, the following reagents were used: low passaged HEK293FT cells (cat. R70007; Thermo Fisher Scientific); T175 flasks (Denville Scientific); Ultraculture media (cat. 12-725F; Lonza) supplemented with 2 mM of L-glutamine, 100 IU/mL of penicillin, 100 μg/ml streptomycin, 1 mM of sodium pyruvate, and 50 mM of sodium butyrate; human T-cell growth medium (X-Vivo 15; Lonza) supplemented with 10 mM of N-acetyl L-Cysteine (cat. A9165; Sigma–Aldrich), 5% human AB serum (cat. HP1022; Valley Biomedical); 55 μM of 2-mercaptoethanol (cat. 31350010; Thermo Fisher Scientific); and IL-2 (50 IU/mL; NCI BRB Preclinical Repository); 10PSGN media prepared with 10% fetal bovine serum and supplemented with 1 × pen/strep, 1 × glutamine, 1 × sodium pyruvate; the UPSGBN is made of Ultraculture media supplemented with 1 × pen/strep, 1 × glutamine, 1 × sodium butyrate, 1 × sodium pyruvate; polyethylenimine (PEI; Sigma–Aldrich); NaCl: 0.15 M filter sterilized; Sucrose (20%) filter sterilized; rectronectin (cat. T100B; Clontech); human T-activator beads, CD3/CD28 Dynabeads (cat. 11132D; Thermo Fisher Scientific); and non-TC treated six-well plates (Denville Scientific).

Plasmids and retroviral packaging system

The following viral packaging plasmids were used: pMD2.G encoding VSV-G pseudotyped envelope protein (cat. 12259; Addgene), pCMVR8.74 (cat. 22036; Addgene), and pAdv (Promega). The pHR_SFFV vector (cat. 79120; Adgene) was used for transgene expression.

Equipment List

A cell culture incubator

Beckman ultracentrifuge

–80°C deep freezer

BSL2 certified hood

Attune NxT Flow Cytometer (Thermo Fisher Scientific)

Automated cell counter (Life Technologies)

Centrifuges

Material

Reagents	Supplier	Specific handling	Storage condition
293FT cells	Thermo Fisher Scientific	Not hazardous. No special handling advice from manufacturer. Always wear personal protective equipment.	LN
Ultraculture medium	Lonza	Not hazardous. No special handling advice from manufacturer.	4°C
L-glutamine	Thermo Fisher Scientific	Not hazardous. No special handling advice from manufacturer.	4°C
Penicillin streptomycin solution	cellgro	Hazardous. Handle with adequate precaution. Always wear personal protective equipment.	−20°C
1 × PBS	Thermo Fisher Scientific	Not hazardous.	4°C
Sodium pyruvate	Thermo Fisher Scientific	Not hazardous. No special handling advice from manufacturer.	4°C
Sodium butyrate	Sigma–Aldrich	Hazardous chemical. Handle with adequate precaution. Always wear personal protective equipment.	Room temperature
N-acetyl L-Cysteine	Sigma–Aldrich	Not hazardous. No special handling advice from manufacturer. Always wear personal protective equipment.	4°C
Human AB serum (heat inactivated)	Valley Biomedical	Serum products should be treated as potentially contaminated. Handle with adequate precaution. Always wear personal protective equipment.	−20°C; in small aliquots at 4°C to avoid repeat freeze–thaw
2-Mercaptoethanol	Thermo Fisher Scientific	Hazardous; open in fume hood. Handle with adequate precaution. Always wear personal protective equipment.	Specially designated chemical safety cabinets
IL-2	NCI-BRB Preclinical Repository	No special handling advice from the supplier.	−20°C in small aliquots to avoid repeat freeze–thaw and bioactivity
Polyethylenimine	Sigma–Aldrich	Health and environmental hazard. Handle with adequate precaution. Always wear personal protective equipment.	Specially designated chemical safety cabinets
NaCl	Sigma–Aldrich	Not hazardous. No special handling advice from the supplier.	Room temperature
Sucrose	Sigma–Aldrich	Not hazardous. No special handling advice from the supplier.	Room temperature
Rectronectin	Clontech	Not hazardous. No special handling advice from the manufacturer.	−20°C in small aliquots to avoid repeat freeze–thaw
CD3/CD28 Dynabeads	Thermo Fisher Scientific	Not hazardous. No special handling advice from the manufacturer.	4°C
pMD2.G	Addgene	Institutional biosafety guidelines must be followed. Must be handled at BSL-2/2+ facility when working to produce virus.	−20°C
pCMVR8.74	Addgene	Institutional biosafety guidelines must be followed. Must be handled at BSL-2/2+ facility when working to produce virus.	−20°C
pHR_SFFV	Addgene	Institutional biosafety guidelines must be followed. Must be handled at BSL-2/2+ facility when working to produce virus.	−20°C
pAdv	Promega	Institutional biosafety guidelines must be followed. Must be handled at BSL-2/2+ facility when working to produce virus.	−20°C

Troubleshooting

Lower titer (section “HER-CAR lentivirus production”). It is essential not to overgrow 293FT cells, which usually results in a lower titer. In addition, collecting virus supernatants in a 32°C incubator can also improve virus titer, as viral RNA is likely to be more stable at this temperature than the usual 37°C.

Storage-related loss of viral titer (section “Virus concentration”). It is essential that viral supernatants are stored at −80°C to avoid loss of viral titer. Once thawed, viral supernatants can remain viable for 1 week at −4°C and must not be refrozen. After 1 week at −4°C, viral supernatant must not be used to transduce T cells.

Lower expression of CAR on T cells (section “Isolation of primary human T cells and bead activation”). Apart from the quality of viral supernatants, contamination of T cells can seriously reduce the expression of CARs. Processing of human blood collars/filters usually acquired from a blood bank is prone to contamination. Therefore, during isolation of PBMCs and subsequent enrichment of CD4 T cells, avoiding contamination of the culture is essential. Contamination seriously affects T-cell activation and expansion, leading to lower expression of CARs. Therefore, T-cell culture must be routinely tested for contamination under a microscope.

Footnotes

Acknowledgments

We deeply acknowledge Dr. Richard Junghans of Tufts Medical Center for the research guidance, lab space, reagents, and assistance in laboratory experiments. We also deeply acknowledge Dr. Wilson Wong of the Department of biomedical engineering Boston University for providing lab space, valuable reagents, and assistance in laboratory experiments. We thank Drs. Jang Hwan Cho and Atsushi Okuma Department of biomedical engineering Boston University for help with virus production and flow cytometry.

Author Disclosure

The authors declare no conflict of interest.

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