CAR-T cell therapy has changed what is possible in cancer treatment. For certain blood cancers, it has delivered remission rates that no other therapy has matched.
Before any CAR-T product reaches a patient, it goes through an intensive preclinical testing phase. The cell lines used in that phase are the foundation everything is built on.
Choosing the right target cell line is not a minor decision. It determines whether your cytotoxicity data is meaningful, whether your antigen targeting is validated, and whether your results will hold when you move to animal models or early clinical trials.
This article covers the cell lines most used in CAR-T preclinical testing, organized by cancer type, with a clear explanation of what each one contributes and why researchers consistently choose it.
What CAR-T Cell Therapy Is and Why Preclinical Models Matter
CAR-T therapy works by engineering a patient's T cells to express a chimeric antigen receptor (CAR), a synthetic protein that directs the T cell to recognize and destroy cancer cells carrying a specific surface antigen.
The preclinical phase answers several critical questions. Does the CAR construct bind its target antigen? Do the CAR-T cells kill tumor cells at relevant ratios? Is there antigen-specific killing or off-target activity?
Cell lines serve as the tumor target throughout these experiments. The line you pick must express the target antigen at levels comparable to patient tumors and produce results that translate into clinically meaningful predictions.
CAR-T Preclinical Cell Lines for B Cell Malignancies
The earliest CAR-T therapies target CD19, expressed across most B cell cancers. CD19-directed constructs were the first approved by the FDA and remain the most studied in the field.
Raji — The CD19 and CD20 Positive Burkitt Lymphoma Standard
Raji is derived from a Burkitt lymphoma patient and expresses high levels of CD19 and CD20, making it the go-to model for evaluating anti-CD19 and anti-CD20 CAR constructs.
It grows rapidly in suspension, is easy to maintain, and produces clean, reproducible killing data in co-culture cytotoxicity assays.
Raji is also commonly used in engineered form with stable GFP or luciferase expression, allowing real-time tracking of tumor cell killing in both in vitro and in vivo experiments.
One consideration: Raji cells are aggressive and disseminate quickly in xenograft models, which makes them useful for modeling advanced disease but requires careful experimental design.
Target antigens expressed: CD19, CD20, CD22, HLA-DR Primary use: CD19 and CD20 CAR-T validation, cytotoxicity assays, in vivo lymphoma xenograft models
NALM-6 — The B-ALL Reference Model for CD19 Targeting
NALM-6 is a B cell acute lymphoblastic leukemia cell line that has become one of the most widely used models for CAR-T preclinical development.
It grows in suspension, expresses robust levels of CD19 and CD22, and has a well-characterized pharmacological profile from decades of published work.
When engrafted into immunodeficient NSG or NKG mice, it establishes consistent leukemia in bone marrow and spleen, mirroring the pattern seen in patients.
Luciferase-expressing NALM-6 variants allow tumor burden to be quantified non-invasively using bioluminescence imaging, making it ideal for tracking CAR-T efficacy in vivo.
Target antigens expressed: CD19, CD22, CD10, CD34 Primary use: Anti-CD19 and anti-CD22 CAR-T efficacy testing, B-ALL xenograft models, bioluminescence-based in vivo tracking
Daudi — The Complement-Sensitive Burkitt Lymphoma Line
Daudi is a Burkitt lymphoma cell line that sees consistent use in CAR-T preclinical work, particularly in studies examining antibody-dependent cellular cytotoxicity alongside direct CAR-T killing.
It is often used in parallel with Raji as part of a two-line lymphoma panel. When a CAR construct kills efficiently in both lines, the finding carries more weight than results from either line alone.
Target antigens expressed: CD19, CD20, CD22, surface immunoglobulin Primary use: CD19 and CD20 CAR-T panels, comparative lymphoma cytotoxicity studies
Jurkat — The CAR Construct Screening Workhorse
Jurkat has an unusual role in CAR-T research: it serves as the effector cell, not the target. It was established from a T cell leukemia patient and expresses TCR-associated signaling machinery.
When a CAR is expressed in Jurkat cells and co-cultured with antigen-positive target cells, upregulation of activation markers like CD69 confirms the CAR is functional and signaling correctly.
This makes Jurkat an efficient, cost-effective first-pass validation step that saves significant time before moving to primary T cells.
Primary use: CAR construct functional validation, T cell activation assays, signaling pathway studies
CAR-T Preclinical Cell Lines for Multiple Myeloma
After the success of CD19-directed therapy, attention turned to multiple myeloma. BCMA (B cell maturation antigen) emerged as the primary target, and several cell lines are now standard models for evaluating BCMA-directed CARs.
MM.1S — The Most Widely Used Myeloma Line
MM.1S expresses high levels of BCMA and SLAMF7 (CS1), two of the most clinically validated targets in myeloma immunotherapy.
It engrafts reliably in NSG and B-NDG mice and works with luciferase expression for bioluminescence tracking of tumor burden.
Several pivotal preclinical studies supporting approved BCMA-targeted therapies used MM.1S as the primary model.
Target antigens expressed: BCMA, SLAMF7 (CS1), CD38, CD138 Primary use: Anti-BCMA CAR-T efficacy and toxicity studies, in vivo myeloma xenograft models, SLAMF7-targeted CAR development
OPM-2 — The High BCMA Expression Myeloma Model
OPM-2 is noted for particularly high surface BCMA expression, making it a sensitive model for evaluating the potency of BCMA-targeting constructs.
It is frequently used alongside MM.1S to cover different antigen density levels and assess whether antigen density affects CAR-T killing efficiency.
Target antigens expressed: BCMA (high), CD38, CD138, CD56 Primary use: BCMA-targeted CAR-T potency testing, high-antigen-density myeloma modeling
CAR-T Cell Lines for AML and Myeloid Malignancies
Extending CAR-T therapy to AML is more difficult because target antigens are also expressed on normal myeloid progenitor cells. Several cell lines are nonetheless standard in AML CAR-T preclinical programs.
THP-1 and U937 — Myeloid Target Models for CD33 and CD123
THP-1 and U937 are the most commonly used myeloid leukemia cell lines for evaluating CAR constructs targeting CD33, CD123, and CLL1.
Both express these antigens at levels relevant to primary AML cells, making them appropriate first-pass models for cytotoxicity screening.
Because myeloid CAR-T therapies face concerns about on-target, off-tumor toxicity, preclinical AML work typically requires parallel testing in normal hematopoietic cells alongside these lines to assess the safety window.
Target antigens expressed (THP-1): CD33, CD123, CD4, CD11b, CD64 Target antigens expressed (U937): CD33, CD64, CD4, CD14 Primary use: AML CAR-T cytotoxicity panels, myeloid antigen targeting, immune evasion research
How to Build a Preclinical Cell Line Panel for CAR-T Research
A single cell line is rarely sufficient to support a CAR-T development program. Regulatory agencies expect evidence of efficacy across biologically relevant models.
A practical CD19-directed panel for B cell malignancies would include NALM-6 for B-ALL coverage and Raji alongside Daudi for lymphoma. Adding a CD19-negative control line such as K562 confirms antigen-specific killing.
For BCMA-directed myeloma programs, MM.1S and OPM-2 together cover different antigen densities. A BCMA-negative control line is essential for confirming specificity.
Building the panel with luciferase-expressing variants from the start enables consistent in vivo tracking and simplifies the transition to xenograft efficacy studies.
Autologous vs Allogeneic CAR-T and the Implications for Cell Line Choice
Most approved CAR-T therapies are autologous, made from the patient's own T cells. Allogeneic, or off-the-shelf, CAR-T therapies from healthy donor T cells are an increasingly active area of development.
Allogeneic programs introduce the complication of potential graft-versus-host reactivity, which expands the safety testing panel. The target cell lines used for efficacy testing remain largely the same.
Cell lines such as MM.1S, NALM-6, and Raji are equally relevant in both autologous and allogeneic CAR-T development.
Frequently Asked Questions
What is CAR-T cell therapy? A cancer immunotherapy where a patient's T cells are engineered to express a receptor targeting cancer cells, then infused back to find and destroy the tumor.
How does CAR-T cell therapy work? The CAR on the modified T cell binds its target antigen on tumor cells, activating the T cell to kill the cancer cell and continue expanding.
What cancers can be treated with CAR-T cell therapy? Approved indications include B-ALL, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma.
What is the success rate of CAR-T cell therapy? In relapsed B-ALL, remission rates reach 70 to 90 percent in clinical trials; in DLBCL, durable remission is seen in roughly 30 to 40 percent of patients long-term.
What are the main side effects of CAR-T cell therapy? The most significant are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), both requiring management in a specialist setting.
What is the difference between autologous and allogeneic CAR-T cell therapy? Autologous uses the patient's own T cells; allogeneic uses healthy donor T cells manufactured in advance for off-the-shelf availability.
What cell lines are most used in CAR-T preclinical research? NALM-6 and Raji for B cell malignancies, MM.1S and OPM-2 for multiple myeloma, Jurkat for CAR construct validation, and THP-1 and U937 for AML programs.
What are the current limitations of CAR-T cell therapy? Key challenges include antigen loss leading to relapse, limited efficacy in solid tumors, high manufacturing cost, CRS and ICANS toxicity, and lengthy autologous production timelines.
Closing Note
The cell lines covered here have been central to almost every major CAR-T therapy that has moved from the lab into patients.
They represent carefully characterized tumor biology that, when used correctly and in combination, generates preclinical data genuinely predictive of clinical outcomes.
Selecting the right lines, building thoughtful panels, and sourcing authenticated cells from reliable suppliers are the practical steps that separate research programs that generate trustworthy data from those that do not.