Considerations for the Development of 

Chimeric Antigen Receptor (CAR) T 

Cell Products

Guidance for Industry

Additional copies of this guidance are available from the Office of Communication, Outreach 

and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 

MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 

from the Internet at https://www.fda.gov/vaccines-blood-biologics/guidance-complianceregulatory-information-biologics.

For questions on the content of this guidance, contact OCOD at the phone numbers or email 

address listed above. 

U.S. Department of Health and Human Services

Food and Drug Administration 

Center for Biologics Evaluation and Research 

January 2024 
Contains Nonbinding Recommendations

i

Table of Contents

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

III. GENERAL CONSIDERATIONS FOR CAR T CELL DESIGN AND 

DEVELOPMENT............................................................................................................. 3

A. CAR Construct...................................................................................................... 3

B. Vector ..................................................................................................................... 3

C. Cellular Starting Material.................................................................................... 4

D. Fresh or Cryopreserved Final Products............................................................. 5

IV. CMC RECOMMENDATIONS....................................................................................... 5

A. Vector Manufacturing and Testing..................................................................... 6

B. Collection, Handling, and Testing of Cellular Starting Material..................... 7

C. CAR T Cell Manufacturing and Testing ............................................................ 8

1. CAR T cell manufacturing process control ...................................................... 9

2. CAR T cell analytical testing.......................................................................... 11

3. Labeling for CAR T cells................................................................................ 15

D. Managing Manufacturing Changes and Assessing Comparability During the 

CAR T Cell Product Lifecycle ........................................................................... 16

1. Change management....................................................................................... 17

2. Comparability study design ............................................................................ 18

E. Single-Site or Multisite CAR T Cell Manufacturing....................................... 19

1. Single-site manufacturing ............................................................................... 19

2. Multisite manufacturing.................................................................................. 19

3. Multisite testing .............................................................................................. 20

V. NONCLINICAL RECOMENDATIONS ..................................................................... 20

A. Nonclinical Considerations for the CAR Construct ........................................ 20

B. Nonclinical Considerations for the Cellular Component of CAR T Cells..... 22

C. In Vivo Testing of CAR T Cells......................................................................... 22

D. CAR T Cells with Additional Modifications .................................................... 23

VI. CLINICAL RECOMMENDATIONS........................................................................... 24

A. Study Population................................................................................................. 24

1. Advanced vs. early disease stage .................................................................... 24

2. Tissue-agnostic approach................................................................................ 24

3. Target identification........................................................................................ 25

4. Pediatric subjects ............................................................................................ 25

B. Treatment Plan.................................................................................................... 26

1. Dose selection, starting dose, and dose escalation.......................................... 26

2. Repeat dosing.................................................................................................. 27

3. Staggering ....................................................................................................... 27

4. Consideration for manufacturing delay or failure........................................... 27
Contains Nonbinding Recommendations

ii

5. Bridging therapy ............................................................................................. 28

C. Clinical Pharmacology Considerations............................................................. 28

1. Pharmacokinetics............................................................................................ 29

2. Pharmacodynamics......................................................................................... 29

3. Immunogenicity .............................................................................................. 30

D. Safety Evaluation and Monitoring .................................................................... 30

1. Clinical monitoring ......................................................................................... 30

2. Toxicity grading.............................................................................................. 31

3. Dose-limiting toxicities (DLTs), stopping rules and attribution..................... 31

E. CAR T Cell Persistence and Long Term Follow-up........................................ 32

F. Allogeneic CAR T Cells...................................................................................... 33

VII. REFERENCES................................................................................................................ 34
Contains Nonbinding Recommendations

1 

Considerations for the Development of Chimeric Antigen Receptor 

(CAR) T Cell Products

Guidance for Industry

This guidance represents the current thinking of the Food and Drug Administration (FDA or 

Agency) on this topic. It does not establish any rights for any person and is not binding on FDA 

or the public. You can use an alternative approach if it satisfies the requirements of the 

applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff 

responsible for this guidance as listed on the title page. 

I. INTRODUCTION

Chimeric antigen receptor (CAR) T cell products are human gene therapy1 products in which the 

T cell specificity is genetically modified to enable recognition of a desired target antigen for 

therapeutic purposes. This guidance is intended to assist sponsors, including industry and 

academic sponsors, developing ex vivo-manufactured CAR T cell products. In this guidance, 

we, FDA, provide CAR T cell-specific recommendations regarding chemistry, manufacturing, 

and control (CMC), pharmacology and toxicology, and design of clinical studies for oncology 

indications (including hematologic malignancies and solid tumors). Recommendations specific 

to autologous or allogeneic CAR T cell products are noted in this guidance. This guidance also 

provides recommendations for analytical comparability studies for CAR T cell products. While 

this guidance specifically focuses on CAR T cell products, some of the information and 

recommendations provided may also be applicable to other genetically modified lymphocyte 

products, such as CAR Natural Killer (NK) cells or T cell receptor (TCR)-modified T cells. 

These related product types can be highly specialized, and in many cases, considerations beyond 

those recommended in this guidance would depend on the specific product and manufacturing 

process. To discuss considerations specific to these related products or non-oncology 

indications, we recommend sponsors communicate with the Office of Therapeutic Products 

(OTP) in the Center for Biologics Evaluation and Research (CBER) before submitting an 

Investigational New Drug Application (IND) (e.g., by requesting a pre-IND meeting (Ref. 1)). 

1 Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of 

living cells for therapeutic use. FDA generally considers human gene therapy products to include all products that 

mediate their effects by transcription or translation of transferred genetic material, or by specifically altering host 

(human) genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified 

microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, 

and ex vivo genetically modified human cells. Gene therapy products meet the definition of “biological product” in 

section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the 

prevention, treatment, or cure of a disease or condition of human beings (see Federal Register Notice: Application 

of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products (58 FR 

53248, October 14, 1993), https://www.fda.gov/media/76647/download).
Contains Nonbinding Recommendations

2

In general, FDA’s guidance documents, including this guidance, do not establish legally 

enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a 

topic and should be viewed only as recommendations, unless specific regulatory or statutory 

requirements are cited. The use of the word should in Agency guidance means that something is 

suggested or recommended, but not required. 

II. BACKGROUND

CAR T cells2 are gene therapy (GT) products that are regulated under FDA’s existing framework 

for biological products. We recognize that the development, manufacture, testing, and clinical 

assessment of CAR T cells is challenging. Careful design and appropriate testing of the CAR 

transgene3 and delivery vector are critical to product safety, specificity, and function. CAR T 

cell manufacturing involves multiple biological materials and complex multi-step procedures, 

which are potential sources of variability among product lots. Thus, control of the 

manufacturing process and appropriate in-process and lot release testing are crucial to ensure 

CAR T cell safety, quality, and lot-to-lot consistency. In addition, changes to the manufacturing 

process are common during product development. It is essential to understand the effects of such 

changes on product quality. Comprehensive product characterization studies are valuable for 

identifying relevant critical quality attributes (CQAs) that can be assessed during manufacture, at 

lot release, and in comparability and stability studies to assure safety and efficacy (Ref. 2).

Critical process parameters (CPPs) can then be established through process qualification, to 

ensure consistent CQAs for every manufactured batch. (Ref. 2). FDA’s guidance entitled

“Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy 

Investigational New Drug Applications (INDs): Guidance for Industry,” January 2020 (Ref. 3)

(hereinafter referred to as the “GT CMC Guidance”) describes the general considerations for GT 

product manufacturing and testing.

Nonclinical evaluation of CAR T cells is necessary to support a conclusion that it is reasonably 

safe to administer the product in a clinical investigation (Title 21 of the Code of Federal 

Regulations 312.23(a)(8) (21 CFR 312.23(a)(8)). Nonclinical testing of CAR T cells can be 

challenging due to the inherent biological complexity and variability of this product type and the 

limited availability of suitable animal models to test safety and activity. A case-by-case 

nonclinical testing strategy should be applied using in vitro, in silico, and in vivo testing 

strategies, as appropriate, in conjunction with available nonclinical and clinical data from related 

products to support use of CAR T cells in a proposed clinical trial.

Well-designed early-phase clinical studies are critical to establish safety of the product, adequacy

of risk mitigation measures, dose-response relationship, differences in optimal dose based on 

differences in indication, preliminary evidence of efficacy, and feasibility of manufacturing. For 

autologous CAR T cells, early-phase studies also provide information on how long it will take to 

manufacture the product and whether bridging therapy will or will not be used as an attempt to 

2 CAR T cell products will be referred to as CAR T cells throughout this guidance.

3 For the purposes of this guidance, transgene means an exogenous gene that is introduced into a host cell. See also 

(Ref. 10).
Contains Nonbinding Recommendations

3

control the active disease while subjects wait for the CAR T cell treatment. For allogeneic CAR 

T cells, early-phase studies can be informative with regards to the risks of graft versus host 

disease (GVHD). Information gained from these early-phase studies support the development of 

CAR T cells in later-phase clinical studies and may expedite the clinical development of CAR T 

cells. 

III. GENERAL CONSIDERATIONS FOR CAR T CELL DESIGN AND 

DEVELOPMENT

CAR T cells are complex products that may incorporate multiple functional elements. The 

nature of these functional elements, how the functional elements are introduced into the cells

(i.e., vector type), the cellular starting material, and the final drug product formulation are all 

critical to product safety, specificity, and function. Here, we briefly outline key considerations 

for CAR T cell design and development. 

A. CAR Construct

CARs generally contain two types of domains: antigen recognition and signaling.

Antigen recognition domains allow CAR T cells to bind to one or more target antigen(s). 

We recommend sponsors assess the ability of each antigen recognition domain to 

specifically bind to its target antigen, as described in section V.B of this guidance. 

Many antigen recognition domains are derived from murine monoclonal antibodies that 

may be immunogenic in humans, leading to rejection of the CAR T cells or other safety 

risks (e.g., anaphylaxis). If approaches to reduce immunogenicity (e.g., “humanization” 

by Complementarity-Determining Region grafting) are used, we recommend the IND 

describe these changes and their impact on target binding and biological activity (Refs. 

4, 5, 6). When multiple CARs are expressed in a single drug product, the CAR construct

design should reduce the risk of recombination events, if feasible.

Signaling domains initiate T cell activation. We recommend that the functionality of 

signaling domains be well supported by information from previous nonclinical and 

clinical experience or thoroughly demonstrated, as described in section V.B of this 

guidance. For example, the contribution of transmembrane domain, hinge, and linker 

regions used to separate different functional regions of the construct should be 

evaluated, as these may affect CAR T cell specificity, persistence, and activity (Refs. 7, 

8, 9). 

B. Vector

A “vector” is a vehicle consisting of, or derived from, biological material that is designed 

to deliver genetic material. Examples of vectors include plasmids, viruses, and bacteria 

that have been modified to transfer genetic material (Ref. 10). For CAR T cells, the 

vector is a critical component that furnishes a pharmacological activity for the treatment 

of disease (section IV.B of the GT CMC Guidance (Ref. 3)). Vectors that integrate into 

cellular DNA (e.g., retroviral-based vectors or transposons) can provide long term 
Contains Nonbinding Recommendations

4

transgene expression compared to non-integrating vectors. Long term follow up is 

recommended for products that include integrating vectors, because integrating vectors 

may increase the risk of delayed adverse events (Ref. 10). The predicted risk of delayed 

adverse events is thought to be low for non-integrating vectors, and generally long term 

follow up would not be needed.

In addition to the CAR, vectors may express additional functional elements. For 

example, vectors may express additional functional elements that allow for the selection 

or enrichment of cellular subsets during manufacturing (Ref. 11); that modify T cell 

persistence and/or activity (Ref. 11); or that allow selective in vivo ablation (“suicide 

genes”) of CAR T cells (Refs. 12, 13, 14).

It should be noted that each additional functional element may affect CAR T cell safety 

and effectiveness. We recommend sponsors provide justification and relevant data to 

support incorporation of additional elements. The justification should include an 

assessment of any impact that these additional elements will have on CAR T cell

specificity, functionality, immunogenicity, or safety (see section V.E of this guidance).

Transgene sequences that are unnecessary for the biological function of a product may 

be immunogenic in vivo or have other unanticipated effects on product persistence or 

activity. As a general guiding principle, we recommend that unnecessary transgenes

(e.g., antibiotic resistance genes used for plasmid selection) should not be included in the 

vector. 

C. Cellular Starting Material 

The starting material for CAR T cell manufacture is generally obtained by leukapheresis

of patients (for autologous products) or healthy donors (for allogeneic products). Safety 

and regulatory considerations differ for autologous and allogeneic products, as outlined in 

section IV.B of this guidance.

4

Particular consideration should be given to patients who have received CAR T cells 

previously. Such patients may be considered for different CAR T cell clinical studies due 

to lack of response to the previously administered CAR T cells, relapse of the same 

condition, or treatment for a different malignancy. CAR T cells manufactured using 

cellular starting material (e.g., leukapheresis) from patients who have received CAR T 

cells previously may differ from the same type of CAR T cells manufactured using 

cellular starting material from patients who have not. Previously administered CAR T

cells in the starting material may have unexpected effects on CAR T cell manufacturing 

(e.g., expansion or transduction rates), potency, in vivo expansion, safety, and efficacy. 

Therefore, evaluation of the previously administered CAR T cell levels in the cellular 

starting material may be appropriate. This may be accomplished by detection of common 

vector or CAR features to evaluate the presence of previously administered CAR T cells. 

In addition, we recommend you collect retention samples of leukapheresis material in the 

4 See also FDA’s guidance entitled “Human Gene Therapy Products Incorporating Human Genome Editing: 

Guidance for Industry,” January 2024 (GE Guidance) (Ref. 15).
Contains Nonbinding Recommendations

5 

event that additional analysis is necessary. If an autologous CAR T cell clinical study 

will enroll patients who have received CAR T cells previously and patients who have not, 

the potential differences in the CAR T cells should be evaluated and considered in the 

clinical study design and analysis. We recommend sponsors discuss these considerations 

for product characterization, testing, dosing, and clinical study design with OTP prior to 

the IND submission as part of a pre-IND meeting (Ref. 1). 

D. Fresh or Cryopreserved Final Products

CAR T cells may be formulated for fresh infusion or cryopreserved for later

administration. The choice of formulation depends on the product development strategy 

and practical constraints. 

Fresh CAR T cells have a limited shelf life before product quality degrades. We 

recommend that the maximum time between formulation and infusion be defined and 

supported by stability studies and include considerations for preparation prior to 

administration. Additionally, the timeframe in which release tests can be performed is 

limited. Therefore, it is crucial to develop and implement well-designed logistics, which 

may include: timing for sampling and testing for lot release; reporting Quality Control 

(QC) testing results and Quality Assurance (QA) review for lot release; scheduling 

product shipping; and receiving and handling of the fresh product at the clinical site. 

On the other hand, cryopreservation allows sufficient time for full release testing and 

flexibility in scheduling patients for infusion. We generally recommend cryopreservation 

when CAR T cells are manufactured at a central location and shipped to clinical sites for 

administration. For cryopreserved CAR T cells, the risks associated with infusion of the 

cryoprotectant should be assessed, and controlled thawing of the product at the clinical 

site may be critical to maintain product quality. 

Regardless of the formulation, there should be appropriate procedures to ensure adequate 

control of the CAR T cells during shipping to the clinical site. These procedures should 

be described in the IND and in place before initiating clinical studies. The procedures to 

ensure CAR T cell product quality during shipping, receipt, storage, and preparation for 

infusion should be validated prior to licensure. 

IV. CMC RECOMMENDATIONS

This section of the guidance addresses considerations specific to CAR T cell products and is not 

designed to be a stand-alone CMC guidance. Please refer to the general CMC guidance 

documents on cell and gene therapies available from FDA’s website: 

https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapyguidances. 

We recommend sponsors organize information in the Common Technical Document (CTD) 

format with the vector CMC information described in a complete Drug Substance (DS) section 
Contains Nonbinding Recommendations

6

and the CAR T cell information organized into a separate DS section and a separate Drug 

Product (DP) section, as discussed in section IV.B of the GT CMC Guidance (Ref. 3). When 

CAR T cells are manufactured using a continuous process where there is no clear division 

between the DS and DP, we recommend that you provide an explanation to support your DS/DP 

distinction in the summary information in Module 2 of the CTD submission. The CTD DS 

sections should follow the format and numbering scheme recommended in Module 3 of FDA’s

Guidance for Industry: “M4Q: The CTD – Quality,” August 2001 (Ref. 16), and the sections 

should be distinguished from one another by including the DS name and manufacturer in the 

heading (e.g., section 3.2.S.1 General Information [name, manufacturer]).

The emphasis for CMC in all phases of development is product safety and manufacturing 

control. We recommend that CAR T cells be developed following a lifecycle approach where 

information is gathered over the course of product development and submitted in a stageappropriate manner. The amount of CMC information to be submitted in your IND depends on 

the phase and the scope of the clinical investigation proposed (21 CFR 312.23(a)(7)). Therefore, 

you may not need to complete all CTD sections in your original IND submission. Similarly, 

manufacturing must comply with Current Good Manufacturing Practice (CGMP), as appropriate 

for the stage of development (section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act 

(FD&C Act) (21 U.S.C. 351(a)(2)(B)) (see also Ref. 17, and 21 CFR 210.2). Additional CMC 

information may be needed to align product development with the clinical development, 

especially when the latter is rapidly progressing under an expedited development program. For 

example, analytical assays should be fit for purpose to support early phase studies and qualified 

before initiating clinical studies that are intended to provide the primary evidence of 

effectiveness to support a marketing application.

For CAR T cells in the early stages of clinical development, very few specifications are 

finalized, and some tests may still be under development (section V.A.4.a of the GT CMC 

Guidance (Ref. 3)). Characterization data collected during early studies can inform release 

criteria used in later development to ensure product and process consistency. Thus, 

characterization studies are crucial to support product development and comparability 

assessments. For studies in which a primary objective is to gather meaningful data about product 

efficacy, we recommend that acceptance criteria be refined to ensure batches are well-defined 

and consistently manufactured. In the Biologics License Application (BLA), the proposed 

commercial lot release criteria should be based on data from product lots shown to be safe and 

effective in clinical studies.

A. Vector Manufacturing and Testing

The GT CMC Guidance (Ref. 3) provides recommendations for manufacturing and 

testing of the vector. The vector safety and quality should be sufficiently characterized 

prior to initiation of clinical studies. For later phase studies and for licensure, the vector

must be manufactured according to CGMP under section 501(a)(2)(B) of the FD&C Act, 

and analytical assays should be validated. (Ref. 18). During CAR T cell BLA review, 

vector manufacturing facilities are subject to inspection.
Contains Nonbinding Recommendations

7

Vector quality directly contributes to the quality and consistency of the CAR T cells. We 

recommend that sponsors describe the vector structure, characterization and testing of the 

Master and Working Cell Banks, characterization of reference materials, and vector 

manufacture and testing. We also recommend stability studies for vectors be conducted 

to support hold and storage times as described in section V.A.7 of the GT CMC guidance 

(Ref. 3). Vector lot release testing should include measures of safety, identity, purity, 

and biological activity. An assay that assesses the biological activity of the transgene 

may be developed in coordination with the CAR T cell potency assay (see section IV.C.2

of this guidance). Transgene expression alone as a measure of biological activity may be 

sufficient to support early-phase IND studies; however, additional measures of biological 

activity will likely be requested for clinical study(s) intended to provide primary evidence 

of effectiveness to support a marketing application. Additionally, we recommend that 

vector strength be determined during lot release testing to normalize the amount of vector 

used for transduction during CAR T cell manufacturing. For example, we recommend

testing viral vectors for transducing units per milliliter (mL) in a suitable cell line or 

healthy donor cells. This allows determination of the amount of vector that is added per 

cell to achieve the target percentage of CAR-positive cells in the CAR T cell DP while 

ensuring that the vector copy number remains within target specifications.

Vector safety testing should include microbiological testing such as sterility, 

mycoplasma, endotoxin, and adventitious agent testing to ensure that the CAR T cell DP 

is not contaminated. Additional testing may be recommended depending on the type of 

transgene vector being used. For example, there are additional safety concerns and 

testing expectations related to the use of retroviral-based vectors (section V.A.4.b.ii of the 

GT CMC Guidance (Refs. 3 and 19)). The recommendations for long term follow-up of 

patients generally depends on the safety concerns associated with the vector and the 

propensity for the vector to integrate (Ref. 10).

B. Collection, Handling, and Testing of Cellular Starting Material 

Here, we describe considerations for cellular starting material, using starting material 

obtained from leukapheresis (referred to as “leukapheresis starting material”) as an 

example. The recommendations in this section may be applicable to other types of 

cellular starting material as well. Testing recommendations for cell banks originating 

from allogeneic cells or tissues are discussed in section V.A.2.c.ii.b of the GT CMC 

Guidance (Ref. 3).

Collection of the leukapheresis starting material should be conducted in accordance with 

the regulations in 21 CFR part 1271. Autologous leukapheresis starting material does not 

require a donor eligibility determination (21 CFR 1271.90(a)(1) and Ref. 20), but you 

may consider a risk-based approach for screening or testing (Ref. 3). Allogeneic 

leukapheresis starting material, on the other hand, does require a donor eligibility 

determination, including screening and testing for relevant communicable disease agents 

and diseases (21 CFR part 1271, Subpart C). 
Contains Nonbinding Recommendations

8

We recommend that procedures used for handling the leukapheresis starting material 

from collection to the start of the manufacturing process are described in the IND as 

discussed in section V.A.2.c.ii of the GT CMC Guidance (Ref. 3). This description 

should include any wash steps or cryopreservation procedures. We recommend these 

procedures, including handling of the cells and shipment to the manufacturing site, be in 

place at all leukapheresis collection sites to ensure quality of the material. You should 

have appropriate procedures in place to ensure adequate control of the leukapheresis 

starting material during shipping to the manufacturing facility (e.g., temperature control), 

and information regarding shipping containers and temperature monitoring should be 

provided. Validation of the shipping process and any hold or cryopreservation steps,

including assessment of leukapheresis starting material stability under the intended 

conditions, should be included for licensure. Once the leukapheresis starting material has 

been received by the manufacturing facility, subsequent manufacturing must comply with

CGMP as appropriate for the stage of development (see Ref. 17, and 21 CFR 210.2). 

Due to patient or donor variability, the cellular starting material can represent a major 

source of lot-to-lot variability in CAR T cell quality and function. The probability of 

manufacturing success may be increased by establishing acceptance criteria for the 

leukapheresis starting material used in CAR T cell manufacturing, as experience is 

gained throughout product development. For example, you may specify a minimum cell 

number, viability, and percent CD3+ cells. To aid in manufacturing failure 

investigations, we recommend that you test the leukapheresis starting material for 

microbial contamination (e.g., sterility or bioburden) prior to initiating CAR T cell 

manufacturing or that you retain a sample for post hoc testing in the event of a DP 

sterility test failure. Additional characterization of the leukapheresis starting material 

(e.g., for percent and absolute number of CD4+ and CD8+ T cells, NK cells, monocytes, 

B cells) may inform the CAR T cell manufacturing process as these characteristics may 

influence T cell selection and expansion and final CAR T cell quality (Refs. 21, 22, 23). 

To maintain the Chain of Identity (COI), labeling and tracking of material, from 

collection all the way through CAR T cell administration, must be documented (21 CFR 

Part 1271 Subpart D). Additionally, for autologous products, we recommend labeling 

include at least two unique identifiers with label checks built into the batch record prior 

to each processing step. The COI should also be maintained at the clinical site with two 

independent patient and label checks at bedside. Please refer to section IV.C.3 of this 

guidance for considerations regarding labeling for the CAR T cell DP.

C. CAR T Cell Manufacturing and Testing

CAR T cell manufacturing is a complex process that should be developed to achieve the 

target product profile (Refs. 24, 25). Recommendations for the manufacture of ex vivo

modified cells, including CAR T cells, are noted in the GT CMC Guidance (Ref. 3). We 

suggest sponsors consider the recommendations in the GT CMC Guidance (Ref. 3), as 

applicable, for: early product characterization (section IV.A); characterization of 

impurities (sections V.A.3.b.i and ii); manufacturing process development (sections 

V.A.2.f and V.B.2.c); and facility considerations (section V.C.1). This guidance provides 
Contains Nonbinding Recommendations

9

specific recommendations and additional details for CAR T cell manufacturing and 

testing.

1. CAR T cell manufacturing process control

Coupled with donor-to-donor variability inherent to the cellular starting material, 

multi-step manufacturing processes can be a source of variability. To minimize 

variability and promote consistency between CAR T cell lots, we recommend the 

manufacturing process be well-controlled. This can be achieved via the use of 

quality materials, in-process control of PPs, in-process testing, and testing of 

intermediates and the final product for CQAs, as appropriate for the phase of 

product development (Ref. 26).

CAR T cell manufacturing often requires specialized reagents, including selection 

reagents, activation reagents, antibodies, cytokines, serum, and growth factors. 

The safety and quality of such materials can vary widely depending on factors 

such as source or vendors. For example, we recommend that human or animalderived components are not sourced from geographical areas of concern for 

potential viral and/or transmissible spongiform encephalopathy (TSE) agent

contamination and that components be tested appropriately for adventitious 

agents. Lot-to-lot variability and stability of reagents can also be a potential risk 

to CAR T cell product quality and safety. We recommend sponsors qualify 

reagents for quality, safety, and identity through vendor qualification programs

and incoming material qualification programs, including quarantine, Certificate of 

Analysis (COA) and Certificate of Origin (COO) assessment, visual inspection, 

and testing, as appropriate according to a risk assessment (Ref. 3).

To assure product safety, CAR T cells should be free of viable contaminating 

microorganisms; however, the final DP cannot be sterilized by filtration or 

terminally sterilized because cells need to be fully viable and functional. 

Therefore, manufacturing should be conducted by using qualified aseptic 

processing under CGMP requirements (Refs. 17, 27), and aseptic processing must

be validated for licensure (21 CFR 211.113, Ref. 28). Product safety is further 

supported by the use of sterility testing (21 CFR 610.12) per United States 

Pharmacopeia (USP) Chapter 71 or an appropriately validated alternative test 

method per USP <1223>.

The IND should contain information documenting the ability to produce CAR T 

cells according to the proposed manufacturing process through the production of 

developmental or engineering batches. To support process development, sponsors 

may cross reference information from highly-related CAR T cell manufacturing

(e.g., same manufacturing process but with a different CAR construct) at the same 

facility. Generally, starting material from a healthy donor is appropriate for 

manufacturing process developmental batches. However, patient-derived starting 

material may have intrinsic properties that affect CAR T cell manufacturing

because of disease state, prior treatment, or other inherent patient characteristics.

Therefore, in some cases, additional manufacturing process development using 
Contains Nonbinding Recommendations

10

patient-derived starting material may be recommended for autologous CAR T 

cells.

We do not require use of approved or cleared medical devices as equipment in 

CAR T cell manufacturing after collection of the leukapheresis starting material. 

Manufacturing equipment (such as centrifugation/washing, selection, or 

incubation equipment, including automated equipment) should be qualified as 

suitable for the intended use. This qualification is the responsibility of the IND 

sponsor, not the medical device or equipment manufacturer. Manufacturing 

equipment operating parameters should be validated to support the BLA.

If information describing reagents5, the vector, manufacturing equipment, 

manufacturing process, or a manufacturing facility has already been submitted to 

the FDA (e.g., in another IND, investigational device exemption (IDE), or Master

File (MF)), a letter from the file holder authorizing FDA to cross-reference the 

previous submission for CMC or other information may be submitted to support 

an IND. Sponsors should specify what information is cross-referenced and where 

the information is contained in the cross-referenced file. Any DS, DS

intermediate, and DP information should be included in the BLA and should not

be incorporated by reference to a MF. Specific questions regarding information 

that should be included in the BLA should be discussed during a pre-BLA 

meeting with FDA.

As experience is gained through product development, CPPs should be identified 

and used to establish in-process controls. Examples include:

• Using a fixed bead: cell ratio at the activation stage.

• Using a constant amount of vector per cell (e.g., a fixed multiplicity of 

infection for viral vectors) and a fixed duration at the gene transfer 

step.

• Using fixed electroporation settings.

• Monitoring cell expansion in culture and maintaining an optimal cell 

density by addition of media.

Appropriate in-process testing at relevant time points is vital to achieve and 

maintain control of the manufacturing process. In-process testing regimens for 

CAR T cells typically assess multiple parameters (e.g., viability, cell number, cell 

phenotype, CAR expression). Results from in-process tests can be used to guide 

manufacturing decisions at critical steps, such as when to change culture media or 

when the CAR T cells are ready to harvest.

5 For the purposes of this guidance, reagents are those materials used for manufacturing (e.g., cell growth, 

differentiation, selection, purification, or other critical manufacturing steps) that are not intended to be part of the 

final product. See also section V.A.2.c.i of the GT CMC guidance (Ref. 3).
Contains Nonbinding Recommendations

11

We recommend stability studies for CAR T cells be conducted to support hold 

and storage times as described in sections V.A.7 and V.B.8 of the GT CMC 

guidance (Ref. 3). In-use stability studies should be conducted to support the 

intended hold time between final formulation and administration for fresh 

products, or the time between thaw and administration for cryopreserved 

products. Products manufactured from healthy donor material may be sufficient 

to support early stability studies. However, for licensure, additional stability 

studies including products manufactured from patient-derived starting material 

should be provided to support the determination of product shelf life. 

2. CAR T cell analytical testing

Analytical testing of CAR T cells is necessary to assure product identity, quality, 

purity, and strength6 of the investigational product (21 CFR 312.23(a)(7)(i)). 

Sections V.A.4 and V.B.5 of the GT CMC Guidance (Ref. 3) provide general 

recommendations on analytical testing of the DS and DP. Section V.A.4.b.i of the 

GT CMC Guidance (Ref. 3) includes recommendations for alternatives to

compendial methods (e.g., for sterility and mycoplasma). Section V.B.5.b.i of the 

GT CMC Guidance (Ref. 3) includes recommendations for cellular products that 

are administered fresh, or with limited hold time between final formulation and 

patient administration.

For allogeneic CAR T cells, where each product lot is meant to treat multiple 

patients, additional testing beyond what is described in this section may be

appropriate. For example, additional adventitious agent testing, stringent 

acceptance criteria for the number of potentially alloreactive lymphocytes, and 

absence of aberrant growth should be included in lot release testing. Additional 

recommendations on analytical testing of CAR T cells incorporating genome 

editing can be found in FDA’s guidance entitled “Human Gene Therapy Products 

Incorporating Human Genome Editing,” (Ref. 15) (hereinafter referred to as the 

“GE Guidance”).

Analytical testing for CAR T cells often requires complex assays and 

development of product-specific biological assays. Thus, we recommend that 

sponsors begin assay development in early stages of CAR T cell development and 

use a variety of assays to characterize their product. In general, scientifically 

sound principles for assay performance should be applied (i.e., tests should be 

specific, sensitive, and reliable and include appropriate controls or standards).

We recommend compendial methods be used when appropriate, and safety6 For purposes of this guidance, “strength” is the equivalent of “potency.” As defined in 21 CFR 600.3(s), the word 

potency is interpreted to mean the specific ability or capacity of the product, as indicated by appropriate laboratory 

tests or by adequately controlled clinical data obtained through the administration of the product in the manner 

intended, to effect a given result. During the IND stage, sponsors must submit data to assure the identity, quality, 

purity and strength (21 CFR 312.23(a)(7)(i)) as well as stability (21 CFR 312.23(a)(7)(ii)) of products used during 

all phases of clinical study. Biological products regulated under section 351 of the PHS Act must meet prescribed 

requirements of safety, purity and potency for BLA approval (42 USC 262(a)(2)(C)(i)); (21 CFR 601.2).
Contains Nonbinding Recommendations

12

related tests should be qualified prior to initiation of clinical studies. Although 

validation of analytical procedures is usually not required for IND submissions 

for Phase 1 studies, we recommend providing information showing appropriate 

control of the test methods. Each assay used for lot release or stability testing 

should be qualified prior to initiating studies intended to provide primary 

evidence of effectiveness to support a marketing application, and assays must be 

validated to support a BLA7 (21 CFR 211.165(e)). 

When changing an assay, a risk assessment should be performed to determine 

how the assay change impacts CAR T cell evaluation. If there are major changes 

to assay methodology, we recommend the assay be requalified to ensure that 

assay performance characteristics remain acceptable. If an assay is replaced with 

a new assay that measures the same attribute in the same way (e.g., change to 

another ELISA kit vendor), the assay should be qualified, and a study may be 

requested to demonstrate that the new assay yields results that are equivalent to 

the old assay (Ref. 29). We recommend that these studies include side-by-side 

analysis of the old and new assays using the same test samples. If an assay is 

replaced with a new assay that measures an attribute in a fundamentally different 

way (e.g., potency assay changed from cell killing assay to cytokine release 

assay), the new assay should be qualified, and data to support any associated 

changes to assay acceptance criteria or the impact on stability should be provided.

a. Flow cytometry

Flow cytometry allows assessment of multiple CAR T cell attributes

throughout the manufacturing process (e.g., cell viability, identity, purity, 

strength). 

i. We recommend that the initial IND submission include:

- A description of the assay, including the flow cytometry 

antibody panel and the gating strategy used to define each

cell population detected. Live/dead stain should be 

included in the flow cytometry panel. We recommend that 

information on relevant cell populations in the final 

product, including those not anticipated to have a 

therapeutic effect (e.g., residual tumor cells, if applicable),

be collected. 

7 Each BLA must include a full description of the manufacturing process, including analytical procedures that 

demonstrate the manufactured product meets prescribed standards of identity, quality, safety, purity, and potency 

(21 CFR 601.2(a) and 601.2(c)). Data must be available to establish that the analytical procedures used in testing 

meet proper standards of accuracy, sensitivity, specificity, and reproducibility and are suitable for their intended 

purpose (21 CFR 211.165(e) and 211.194(a)(2)).
Contains Nonbinding Recommendations

13

- A brief summary of instrument calibration and QC

activities (e.g., beads used and frequency of calibration 

runs) to ensure accuracy of the results.

- A list of assay controls. Controls may include: single 

stained compensation controls for calculating compensation 

values; Fluorescence Minus One (FMO) controls to 

determine fluorescence spread and gating boundaries for 

minor populations; and isotype controls to identify 

nonspecific binding. Throughout assay development, 

system suitability specifications for each control should be 

established.

ii. As part of assay development, we recommend you establish 

and implement written procedures to ensure proper sample 

staining, acquisition and data analysis. 

iii. We recommend performing antibody titration to determine the 

optimal antibody dilution. 

iv. We recommend direct detection of the CAR to determine the 

percentage of CAR-positive cells. If the CAR is detected by 

surrogate protein expression (e.g., detection of a co-expressed 

gene) or other broad-specificity reagents (e.g., protein L), you 

should evaluate the correlation with CAR expression.

Assessment of the sensitivity and specificity of the surrogate 

marker should be included as part of the justification for use.

v. A comprehensive validation study for lot release flow 

cytometry assay(s) must be conducted to support licensure.

8

(21 CFR 211.165(e)). This validation study should be 

conducted per International Conference on Harmonisation

(ICH) Q2 (Ref. 30) and include validation of each fluorescently

labeled marker in the flow cytometry panel on the flow 

cytometer(s) used for CAR T cell release. Robustness studies,

including defining the maximum holding time for samples

before staining and between staining and acquisition, should be 

included. 

b. Vector Copy Number (VCN) 

Vector integration can potentially alter expression of cellular genes and 

contribute to tumorigenicity (Refs. 31 and 32). Therefore, vector 

integration in the DP is an important safety attribute to measure for CAR 

T cell release. For integrating vector systems, the average number of 

integrations per CAR-positive cell, generally referred to as VCN, should 

be determined and reported on the Certificate of Analysis (COA) for each 

lot. Determining VCN as a function of total cells includes CAR-negative

cells in the denominator and lowers the reported vector integration rate. 

8 See footnote 6.
Contains Nonbinding Recommendations

14

Using the percentage of CAR-positive cells, the average VCN per CARpositive cell can be calculated. VCN as a function of CAR-positive cells 

will provide a more accurate representation of the VCN in modified cells 

and thus a more accurate representation of product risk for insertional 

mutagenesis. We recommend that the manufacturing process be 

optimized to control VCN while meeting the target CAR-positive cell 

frequency.

We recommend that the VCN release criterion be justified based on a risk 

assessment. The risk assessment may include supporting data from 

studies such as insertion site analysis, clonal dominance, dose, indication, 

study population, etc. Supporting experimental data may be obtained from 

developmental and engineering manufacturing runs.

For CAR T cells manufactured without extended culture, determining the 

stably integrated VCN at the time of lot release testing may be difficult

(e.g., due to persistence of episomal copies of non-integrated vectors). In 

some cases, an interim VCN assessment at the time of lot release,

followed by subsequent VCN assessment(s) on cultured CAR T cells, may 

be needed to determine the stably integrated VCN. The appropriate

duration of extended culture for the stably integrated VCN (and other 

release assays, as applicable) is product-specific and should be determined 

experimentally.

c. Identity 

Identity testing is required at all phases of development (21 CFR 

312.23(a)(7)) and must be performed on the contents of a final container 

of each filling of each lot for licensure (21 CFR 610.14). Identity testing 

should adequately identify a product and distinguish it from other products 

in the same facility. Of note, we recommend that identity testing for CAR 

T cells include an assay to measure the presence of the transgene (e.g., 

CAR expression by flow cytometry, gene detection by PCR) and an assay 

specific for the intended cellular composition of the final product (e.g., T 

cell surface markers) as discussed in section V.B.5.b.ii of the GT CMC 

Guidance (Ref. 3). HLA typing may be performed; however, HLA typing 

does not detect the genetic modification and, therefore, is not a sufficient 

identity test. Additionally, HLA typing will not replace requirements for 

maintaining chain of identity (section IV.B of this guidance).

d. Potency 

The CAR T cell DP must be tested for potency (Ref. 33). Upon antigen 

engagement, CAR T cells kill target cells using multiple mechanisms. 

Therefore, the use of orthogonal methods (e.g., cell killing assay, 

transduction efficiency measurements, and cytokine secretion assays) may 
Contains Nonbinding Recommendations

15

be recommended to measure potency. Characterization of CAR T cell 

function during product development will support comparability studies 

and will allow you to determine the most appropriate assays to use for 

commercial lot release.

If the CAR T cells express multiple transgene elements, each transgene 

may contribute to product safety and efficacy and therefore should be 

adequately controlled. A potency assay to measure the intended biological 

activity of each element may be needed, depending on the contribution of 

each transgene to the product’s activity. Justification for the proposed 

assays may be supported by characterization studies. For example:

- If the CAR T cell targets multiple antigens (e.g., CD19 and 

CD22), you should assess the activity of the CAR T cells 

against each individual target antigen because T cell activation 

upon engagement with either antigen is required for the 

product’s function.

- If the CAR T cell includes a cytokine transgene to enhance the 

CAR activity, you should assess the activity of the CAR T 

cells against the target antigen and production of the transgenic 

cytokine because the cytokine is not primarily responsible for

the CAR T cell activity specific to the target antigen. 

- If the CAR T cell includes a transgene conferring drug 

resistance, you should assess drug resistance and CAR T cell 

activity because they have independent mechanisms of action.

3. Labeling for CAR T cells

Your IND must contain a copy of all labels and labeling to be provided to each 

investigator in the clinical study (21 CFR 312.23(a)(7)(iv)(d)). We recommend 

that you include sample or mock-up labels in Module 1 of the CTD. Please note 

that IND products must bear a label with the statement, “Caution: New 

Drug―Limited by Federal (or United States) law to investigational use” (21 CFR 

312.6). We recommend that the label include the product name, manufacturer 

information, and, as applicable, the warnings “Do not filter” and “Do not 

irradiate”. Labeling for licensed CAR T cells must conform to the requirements 

in 21 CFR Part 201 and 21 CFR Part 610 Subpart G, as well as other applicable 

provisions in the FD&C Act.

9

 

Additional labeling is required for autologous CAR T cells. Specifically, CAR T 

cells manufactured from autologous starting material must be labeled “FOR 

AUTOLOGOUS USE ONLY” (21 CFR 1271.90(c)(1)). The label must state 

“NOT EVALUATED FOR INFECTIOUS SUBSTANCES,” unless you have 

performed all otherwise applicable screening and testing under 21 CFR 1271.75, 

9 See also sections 581 and 582 of the FD&C Act (21 U.S.C. 360eee), as added by the Drug Supply Chain Security 

Act (DSCSA) (Title II of Public Law 113-54).
Contains Nonbinding Recommendations

16 

21 CFR 1271.80, and 21 CFR 1271.85 (21 CFR 1271.90(c)(2)). In addition to 

these requirements, it is recommended that the label include at least two unique 

identifiers to confirm patient identification prior to administration. 

CAR T cells must also be labeled with the Biohazard legend shown in 21 CFR 

1271.3(h), if the results of any screening or testing performed indicate the 

presence of relevant communicable disease agents and/or risk factors for or 

clinical evidence of relevant communicable disease agents or diseases. Labeling 

must also bear the statement “WARNING: Reactive test results for (name of 

disease agent or disease),” in the case of reactive test results (21 CFR 

1271.90(c)(5)). 

D. Managing Manufacturing Changes and Assessing Comparability During the

CAR T Cell Product Lifecycle

 

We recognize there may be changes to the CAR T cell design, manufacturing process, or 

manufacturing facility during product development or post-approval. Changes during the 

CAR T cell product lifecycle, including changes to the final container, cytokines used 

during culture, or duration of cell expansion, may impact product quality, safety, 

efficacy, or stability10. There are some changes (e.g., changes to the CAR construct or 

changing from an autologous to allogeneic product) which would generally result in a 

new product that should be submitted in a new IND (Ref. 34). 

Each change is assessed on a risk-based, case-by-case basis, and we recommend sponsors 

communicate with OTP (e.g., through an IND amendment requesting advice or a formal 

meeting request (Ref.1)) when considering such changes.11 When planning such 

changes, we generally recommend sponsors consider the following: 

• Substantial changes to the vector manufacturing process (e.g., changing 

from adherent to suspension culture) should be supported by 

comparability studies. Due to the essential role of the vector in CAR T 

cell activity, the impact of such changes should be assessed on both the 

vector and the CAR T cells. Studies should include side-by-side analyses

of the pre- and post-change vector. Additionally, CAR T cells 

manufactured with pre- and post-change vector should be assessed using 

side-by-side analysis by using the same cellular starting material (e.g., 

splitting the leukapheresis starting material from the same donor). 

• The complexity of comparability assessments may differ depending on the 

extent of the change to the vector or CAR T cell manufacturing process. 

10 During the investigational phase, some CMC changes without adequate comparability data may result in the trial 

being placed on clinical hold (21 CFR 312.42).

11 See also the Draft Guidance for Industry “Manufacturing Changes and Comparability for Human Cellular and 

Gene Therapy Products,” issued July 2023, available at: https://www.fda.gov/media/170198/download. When 

finalized, this guidance will represent FDA’s current thinking on this topic.
Contains Nonbinding Recommendations

17

For example, a small change in the volume of culture media to 

manufacture CAR T cells may generally be supported by cell viability and 

expansion data. In contrast, a more robust comparability study should be 

conducted for a change to the concentration or type of growth factors or 

supplements in the culture media.

• When the CAR T cells or vector manufacturing facility is changed, 

product comparability from the pre- and post-change manufacturing 

facilities should be assessed.

1. Change management

Prior to implementation of any change, you should conduct a risk assessment to 

evaluate the potential impact of the intended change on product quality and

safety. Understanding the impact of the change is critical to evaluate the ability to 

combine clinical data generated pre- and post-change. This risk assessment 

should be based on empirical data generated using developmental lots not 

intended for administration to patients. This risk assessment should inform

whether an analytical comparability study is warranted. Additionally, the stage of 

product development may impact whether an analytical comparability study is 

warranted. For changes to be implemented during early-stage development, the 

major consideration should be avoiding a negative impact on product safety. 

However, when considering changes to be made at later stages of product 

development, the sponsor should evaluate the impact of the change on both safety 

and efficacy. Depending on the type of change, assessment of product stability 

should also be considered. You must submit changes to the CMC information as 

amendments to the IND (21 CFR 312.31(a)(1)). We recommend that details of 

the proposed change(s), the accompanying risk assessment, and the proposed 

change management strategy be submitted as an amendment to the IND, prior to 

initiation of comparability studies or implementation of the change. 

Analytical comparability of CAR T cells pre- and post-change may be assessed 

following the general principles described in ICH Q5E (Ref. 35). Note that the 

term “comparability” does not necessarily mean that pre- and post-change 

products are identical, but that they are highly similar and that any differences in 

product CQAs have no adverse impact on CAR T cell quality, safety, or efficacy

(Ref. 36). A key function of demonstrating analytical comparability is to ensure 

that the clinical data generated pre-change continues to be relevant to the safety 

and efficacy of the post-change product. If there is insufficient evidence to 

demonstrate analytical comparability, then new nonclinical or clinical studies may 

be requested, potentially delaying product licensure. Before initiating analytical 

comparability studies and data analyses, we recommend that you discuss the study 

design and acceptance criteria with OTP.

In some cases, a change might alter CQAs that cannot be adequately measured in 

analytical assays. In such a case, analytical comparability studies will be 
Contains Nonbinding Recommendations

18

inadequate to evaluate comparability. Therefore, we recommend sponsors 

anticipate changes needed to establish a scalable and robust manufacturing 

process and make those changes prior to initiating clinical studies that are

intended to provide primary evidence of effectiveness to support a marketing 

application.

Regardless of the product development stage, the IND must be updated to reflect 

the change in manufacturing process (a change in manufacturing process would 

be considered new CMC information requiring an information amendment; 21 

CFR 312.31(a)). When changes are introduced during late stages of development,

and there are no plans for additional clinical studies to support a BLA, the 

analytical comparability studies should be as comprehensive and thorough as 

those conducted for a licensed product. Differences in CQAs may warrant new

nonclinical or clinical studies.

For a licensed product, manufacturing changes must take place within the context 

of existing change control procedures (21 CFR Parts 210 and 211 and (Ref. 37)). 

Such procedures should be designed to ensure that manufacturing changes do not 

affect CAR T cell quality. If changes to product release criteria are proposed, 

clinical data generated under an IND may be requested to support the safety and 

efficacy of the post-change product.

2. Comparability study design

The extent of comparability studies will depend on the change, the ability of 

analytical methods to detect changes in the product, and the stage of clinical 

development. We recommend that the comparability study design includes

justification that the proposed assays are appropriate to detect potential effects of 

the change(s) on product safety and efficacy as appropriate for the phase of 

product development. Demonstrating that product manufactured with the 

proposed changes can meet current lot release criteria is typically insufficient to 

establish comparability. Comparability studies should be analyzed using 

appropriate statistical methods and predefined acceptance criteria based on lots 

shown to be safe and effective.

Early product characterization to establish CQAs facilitates the design of

comparability studies. Using a variety of characterization assays throughout CAR 

T cell development provides a greater understanding of the product and supports 

the evaluation of quality attributes that may be affected by proposed 

manufacturing changes. For example, you may propose to change the cytokines 

used for CAR T cell culturing to alter the cell expansion rate. However, this 

change may also affect the cellular subpopulations and activation state. 

Therefore, a variety of product attributes, including cellular surface markers,

should be monitored using reliable analytical methods, in addition to those 

attributes typically tested for lot release.
Contains Nonbinding Recommendations

19

Some CAR T cell attributes are intrinsically linked to attributes of the cellular

starting material. Due to the inherent variability of the cellular starting material

for autologous CAR T cells, using historical lots to assess comparability may not 

be adequate. We recommend that CAR T cell comparability be assessed by 

side-by-side testing using the same cellular starting material, when possible. For 

example, leukapheresis starting material from the same donor can be split into 

two portions and used to manufacture product using the pre-change process with 

the other portion used to manufacture product by the post-change process. In 

some cases, comparability studies may be appropriately conducted using CAR T 

cells derived from healthy donors. Any potential differences between patient and 

healthy donor material should be considered when designing comparability 

studies and interpreting the results. You may consider including patient-derived

starting material to supplement studies using healthy donor material if potential 

differences are identified.

E. Single-Site or Multisite CAR T Cell Manufacturing

1. Single-site manufacturing

CAR T cells may be manufactured at a single, centralized location. In this 

situation, the cellular starting material is collected (e.g., at apheresis centers for 

leukapheresis starting material) and shipped to a centralized manufacturing 

facility where the CAR T cells are manufactured. The CAR T cells are later 

shipped to local or distant clinical site(s) for administration. Single-site 

manufacturing may reduce the potential for product variability arising from 

differences between manufacturing facilities. However, there may be logistical 

concerns with cryopreservation or shipping of the cellular starting material, the 

final CAR T cells, and the test samples.

2. Multisite manufacturing

The same type of CAR T cells may be manufactured at several facilities. 

Multisite manufacturing may shorten the timeline from cellular starting material 

collection to administration for autologous products; however, differences

between manufacturing facilities may contribute to product variability. In this 

case, you should demonstrate that a comparable product is manufactured at each 

location to support the analysis of the clinical trial results. Sponsors should also

demonstrate that analytical methods are comparable across the different sites, if 

applicable. 

As the IND sponsor, it is your responsibility to confirm that each manufacturing 

site is following CGMPs (21 CFR 200.10(b), 21 CFR 211.22(a), section V.2.a of 

the GT CMC guidance (Refs. 3 and 38)). We recommend using the same 

standard operating procedures (SOPs), training, reagents, and equipment across 

manufacturing facilities, when possible. We also recommend that the IND
Contains Nonbinding Recommendations

20

describe any differences in the manufacturing process across the manufacturing 

sites. 

Defined acceptance criteria for product quality attributes will help support 

production of similar products across manufacturing sites. We recommend you 

demonstrate analytical comparability of the products manufactured at each site by 

submitting data from CAR T cells manufactured using the same cellular starting 

material (e.g., splitting the leukapheresis starting material from the same donor). 

You should include a list of the methods used for testing and the predefined 

acceptance criteria used for determining analytical comparability. When 

assessing analytical comparability among multiple manufacturing facilities, we 

recommend that you identify a reference site to which all other sites are 

compared. In addition, demonstration of comparability between product lots

produced at different manufacturing sites is critical if the corresponding clinical 

data are combined for efficacy analyses.

3. Multisite testing

Multisite manufacturing is often associated with the same assay being performed 

at multiple testing sites. For example, flow cytometry is often performed at the 

time of DS harvest and, therefore, may need to be performed at an analytical lab 

associated with each manufacturing facility. In this case, we recommend using an 

assay transfer protocol to ensure that non-compendial testing performed at each 

site is suitable for the intended purpose and is reproducible among all testing sites. 

We recommend that the same SOPs, reference materials, reagents, and equipment 

be used across testing facilities, when possible. When available, standard 

materials should be used to calibrate equipment at multiple sites to support 

instrument harmonization. For compendial assays, reproducibility across testing 

sites generally does not need to be demonstrated; however, it is important to 

verify that each site can perform the test as intended.

V. NONCLINICAL RECOMENDATIONS

The objective of a nonclinical program for an investigational product is to support a conclusion 

that it is reasonably safe to administer the product in a clinical trial. Although the diversity and 

inherent biological properties of GT products, including CAR T cells, necessitate a case-by-case 

testing strategy, general considerations for nonclinical testing have been previously 

communicated (Ref. 39). 

A. Nonclinical Considerations for the CAR Construct 

The design of the CAR construct and the process by which the transgene is delivered to 

the T cells are critical in determining product safety and activity. Genetic material 

encoding the CAR can be delivered to T cells using multiple vector types, such as 

gammaretroviral and lentiviral vectors, transposons, and mRNA (Ref. 10). 
Contains Nonbinding Recommendations

21

A major determinant of CAR T cell safety and efficacy is the antigen recognition domain

used to confer target specificity. The antigen recognition domain may originate from 

monoclonal antibodies (mAbs), endogenous ligand/receptor pairs, or from other sources.

Nonclinical evaluation of the antigen recognition domain should include assessment of 

the specificity and affinity/avidity for the target antigen/cells to evaluate the potential for 

on-target/off -tumor and off-target toxicities. Undesired targeting of healthy/normal 

tissues that express the intended target antigen (on-target/off-tumor), as well as 

unintended targeting of other antigens expressed on healthy/normal tissue is a safety 

concern that may be evaluated using in vitro and/or in vivo studies. Examples include: 

(1) tissue cross-reactivity studies using a monoclonal antibody or fusion protein with the 

same antigen recognition domain; (2) protein arrays; (3) cytotoxicity/cytokine release 

testing on panels of human primary cells, cell lines, or induced pluripotent stem cellderived test systems for various organs/tissues; and (4) relevant animal models12. We 

recommend including information from previous clinical experience with a CAR or 

monoclonal antibody with an identical antigen recognition domain, if available, which 

may reduce or eliminate the recommendation to perform additional specificity and 

affinity testing. Sponsors are encouraged to explore a combination of methods to 

minimize the risk to study subjects and to inform the design of the clinical trial. 

Identification of potential on-target/off-tumor and off-target activity can be invaluable in 

establishing enrollment criteria and specific post-infusion assessments and monitoring 

plans.

Characterization of the target antigen is also recommended. Existing clinical experience 

with the target antigen and the tissue expression profile of the target antigen can provide 

supporting information regarding potential off-tumor targets of the investigational CAR T 

cells. However, antigen recognition domains targeting the same antigen as previous CAR 

T cells may have a different safety profile and present different toxicity risks. Antigen 

recognition domains may vary in their affinity or avidity for the target or recognize a 

different site on the antigen which should be evaluated nonclinically. Additionally, CAR 

T cells and monoclonal antibodies that utilize the same single-chain variable fragment 

(scFv) may differ in their safety profile due to the inherent differences between the 

products (e.g., capacity for CAR T cells to traffic, expand, produce cytokines, induce 

cytotoxicity, and persist). These differences and their impact on product safety should be 

considered when characterizing the safety profile of the CAR T cell product. 

A variety of activation and co-stimulatory domains have been incorporated into CAR T 

cells, including the CD3ζ chain, 4-1BB (CD137), CD28, and CD40. These domains have 

been used in various combinations. Depending on the cell type, certain combinations of 

co-stimulatory domains can lead to different biological properties, such as unique 

12 The nonclinical program for any investigational product should be individualized with respect to scope, 

complexity, and overall design. We support the principles of the “3Rs,” to reduce, refine, and replace animal use in 

testing when feasible. Proposals, with justification for any potential alternative approaches (e.g., in vitro or in silico 

testing), should be submitted during early communication meetings with FDA. We will consider if such an 

alternative method could be used in place of an animal test method.
Contains Nonbinding Recommendations

22

cytokine secretion profiles. This can impact the extent of in vivo cell expansion, 

persistence, and activation of other immune cell types. Addressing the potential for CAR 

T cells to undergo stimulation-independent growth, including cytokine and antigenindependent growth, and uncontrolled proliferation is an important aspect of nonclinical

evaluation. Furthermore, the capacity of CAR T cells to secrete cytokines and mediate 

cytolysis should be antigen-dependent, which can be tested by exposure to various cells 

that vary in their expression of the target antigen. The transmembrane domain and hinge 

regions can also impact CAR T cell safety and activity. These regions may modify the 

on-target activity by affecting the flexibility of the antigen recognition domain and 

impact off-target activation (Ref. 40). Comprehensive assessment and characterization of 

these product characteristics can be accomplished using in vitro and in vivo testing 

approaches to evaluate antigen-dependent and antigen-independent activity.

B. Nonclinical Considerations for the Cellular Component of CAR T Cells

The nature of the transduced T cells expressing the CAR can also influence the biological 

activity of the final investigational product. Examples of various T cell populations used 

to express the CARs include: (1) purified T cell subsets; (2) pools of unselected T cells

containing other contaminant cells (e.g., NK cells, B cells, etc.); (3) T cells specific to 

viral antigens (e.g., cytomegalovirus (CMV), Epstein-Barr virus (EBV)); and (4) selected 

stem-like or “young” T cells. The potential for uncontrolled proliferation and toxicity 

may differ depending on the cell source. Thus, nonclinical evaluation may include 

examination of uncontrolled proliferation, in vitro and in vivo testing for T cell clonality, 

karyotypic analysis, TCR repertoire analysis, and specificity for viral antigens through ex 

vivo stimulation and recognition assays. 

The T cells may also be autologous or derived from allogeneic sources. For allogeneic 

CAR T cells, we recommend providing data to address issues such as the potential for a 

graft versus host response or host rejection of the CAR T cells. Additional nonclinical

testing may be requested if genome editing techniques are used to minimize alloreactivity 

(see section V.E of this guidance).

C. In Vivo Testing of CAR T Cells

Animal models can be useful in demonstrating proof-of-concept data for CAR T cell 

functionality. There are several limitations due to species specificity of the CAR T cells

and the tumor target(s), xenogeneic graft versus host response, as well as the difficulties 

in modeling human immune responses in animals. Despite these limitations, in vivo

testing in murine xenograft models (i.e., human tumor xenograft-bearing mouse models 

administered human CAR T cells) can provide information on the trafficking and 

proliferation profile of CAR T cells. 

If a relevant surrogate product is available, syngeneic tumor animal models can provide 

information regarding the interaction of the surrogate CAR T cells with an intact host 

immune system and potential on-target/off-tumor toxicities. Data should be provided to 

support the suitability of the model, such as the binding affinity of the antigen recognition 
Contains Nonbinding Recommendations

23

domain for the human target versus animal target and the expression profile of the target 

antigen in the species being evaluated. Furthermore, characterization of CAR T cell 

behavior, such as target-dependent activation and proliferation, and anti-tumor responses 

(e.g., tumor size, animal survival) can provide supportive rationale for product testing in 

humans. 

Due to the nature of CAR T cells, which are expected to expand in vivo to varying 

degrees, the selection of a starting dose level is often not determined based solely on 

animal studies. Previous clinical experience with similar CAR T cells can often inform 

the starting dose level, dose escalation plan, and dosing regimen in the study population.

In vitro and in vivo studies to characterize the effector-to-target ratio, kinetics of cell 

expansion, and pharmacodynamic/pharmacokinetic anti-tumor responses may be 

informative to guide dose selection.

D. CAR T Cells with Additional Modifications

CAR T cells can include additional components in the transgene, such as suicide genes, 

detection/selection genes, or immunomodulatory elements. Genome editing or gene 

silencing techniques may also be used to modify the CAR T cells to reduce 

immunogenicity (e.g., for allogeneic CAR T cells) or increase activity or persistence.13

Additional nonclinical testing may be needed for novel accessory molecules and genetic 

modifications to evaluate functionality of the specific elements and safety of the 

investigational product. For example, mixed lymphocyte reactions may be informative to 

evaluate the immunogenicity of products that are modified to reduce the risk of GVHD 

and immune responses against allogeneic products. Additional modifications that affect 

CAR T cell persistence may be assessed by cytokine-independent growth assays or 

appropriately designed in vivo studies. When suicide genes and cell markers/tags are 

incorporated, we recommend conducting nonclinical studies to demonstrate their function

and to establish dosing of any additional drug or biological product that is critical to 

induce CAR T cell depletion. 

The parameters that define CAR T cell safety and activity are multifactorial.

Considerations include: (1) the design of the CAR construct (e.g., antigen recognition

domain, signaling domains, transmembrane and hinge domains); (2) ex vivo vector 

delivery method; (3) T cell source; (4) manipulation processes (e.g., activation, cell 

selection); (5) biological activities (e.g., cytokine expression profiles, cytotoxicity, 

proliferation); (6) addition of novel components (e.g., suicide genes, detection/selection 

genes, immunomodulatory elements, genome editing or gene silencing techniques); and 

(7) route of administration. A combination of multiple testing strategies should be used 

for a comprehensive nonclinical testing program (Ref. 39). This information, along with 

available nonclinical and clinical data for related products, can inform clinical trial design 

and support the administration of investigational CAR T cells to human subjects. 

13 Sponsors may also wish to refer to the nonclinical section (section IV) of FDA’s GE Guidance (Ref. 15) for 

additional nonclinical considerations. 
Contains Nonbinding Recommendations

24

VI. CLINICAL RECOMMENDATIONS

This section describes the clinical considerations for early-phase development of CAR T cells for 

patients with cancer (hematologic malignancies and solid tumors). A primary objective of earlyphase clinical trials should be an assessment of safety. Other objectives may include 

determination of optimal dosage, pharmacokinetic/pharmacodynamic (PK/PD) studies, 

evaluation of clinical activity or efficacy, selecting an appropriate population for further clinical 

studies to investigate efficacy and safety, and other scientific objectives.

A. Study Population

Selection of the study population should consider the anticipated risks and potential 

benefits for the study subjects to ensure that the overall study benefits outweigh the 

risks. 

1. Advanced vs. early disease stage

CAR T cells have been associated with considerable toxicities, notably cytokinerelease syndrome (CRS) and neurological toxicities. In some cases, these 

toxicities can be life-threatening and fatal. Therefore, in defining the study 

population, we recommend you consider these toxicities in the context of the 

potential benefit, disease stage, and other available therapies. 

In early-phase trials, sponsors should consider enrolling subjects with severe or 

advanced disease who have not had an adequate response to available medical 

treatment or who have no acceptable treatment options. If designed to enroll 

these subjects, we recommend the trial include procedures to ensure that each 

subject’s treatment options have been adequately evaluated, and the clinical 

protocol describe the measures to capture the pertinent information regarding 

prior therapies and justification for enrollment of these subjects. 

In subjects who have early-stage disease and available therapies, the unknown 

benefits of first-in-human (FIH) CAR T cells may not justify the risks associated 

with the therapy. For any study, the IND submission should provide your 

rationale and justification for the proposed study population, and the informed 

consent document must describe the reasonably foreseeable risks associated with 

the trial as well as alternative courses of treatment (21 CFR 50.25).

2. Tissue-agnostic approach

CAR T cells target a specific antigen (or antigens) expressed by the cancer 

cell regardless of cancer type. Early-phase trials that include subjects with

different cancer types but share a common target antigen (e.g., tissue-agnostic 

approach) may face challenges in evaluating the efficacy and extent of 

toxicities. The disparities in underlying comorbidities of the subjects, the 
Contains Nonbinding Recommendations

25

impact of pre-existing tumor burden on toxicities, and differences in dose 

response relationship may present challenges to the objectives of an 

early-phase study in evaluating the toxicities and dosing. If you plan to 

develop a product for the treatment of more than one cancer type using a 

tissue-agnostic approach, you may consider an early-phase trial that assigns 

subjects to separate cohorts by the disease types and evaluate the doseresponse relationship and severity of toxicities through parallel doseescalations in these cohorts. We recommend your IND submission includes

your rationale for the proposed study design and analysis.

3. Target identification

The anti-tumor effect of the CAR T cells depends on the binding of the CAR 

with the cognate antigen expressed on the cancer cell. Therefore, it is

essential to enroll patients whose tumors express the antigen targeted by the 

CAR T cells. If a test for the target antigen is not commercially available, a 

companion diagnostic test may need to be developed to appropriately select 

subjects for the study. (Ref. 41). Refer to FDA guidances on using these tests 

for oncology trials, including the streamlined process for study risk 

determination (Ref. 42) and principles for co-development of an in vitro

companion diagnostic device with a therapeutic product (Ref. 43). In these 

situations, we recommend the clinical protocol include a detailed description 

of these tests.

4. Pediatric subjects

Some CAR T cells are developed specifically for pediatric conditions. 

Sponsors who are developing CAR T cells to treat pediatric diseases should 

consider how they will incorporate the additional safeguards for pediatric 

subjects into clinical investigations in the overall development program.

Clinical development programs for pediatric indications usually obtain initial 

safety and tolerability data in adults before beginning studies in children. We 

recognize that in some situations, it may be appropriate to initiate clinical 

studies of CAR T cell products in children, and in such situations, adequate 

justification should be provided in the clinical protocol. Title 21 CFR Part 50,

Subpart D, provides the process for additional safeguards required for 

children in clinical investigations. In addition, see section IV.B.5 of FDA’s 

guidance entitled “Considerations for the Design of Early-Phase Clinical 

Trials of Cellular and Gene Therapy Products; Guidance for Industry,” June 

2017 (Ref. 44) and the draft guidance entitled “Ethical Considerations for 

Clinical Investigations of Medical Products Involving Children

Draft Guidance for Industry, Sponsors, and IRBs,” September 2022 (Ref. 45) 

for additional recommendations on including pediatric subjects in cell and 

gene therapy trials. 
Contains Nonbinding Recommendations

26

B. Treatment Plan

1. Dose selection, starting dose, and dose escalation

a. Dose selection

CAR T cell dose selection is complex, necessitating several factors to be 

considered. 

Transduction efficiency can differ from lot to lot, resulting in variation in 

the percentage of transduced cells. This variation can lead to substantial 

differences in the active cell dose administered to different subjects, even 

when the same total cell dose is administered. Ideally, manufacturers 

should work to control variability in the transduction process. However,

even with a consistent manufacturing process, such variations in 

transduction efficiency are expected to occur. To mitigate this variability 

in dosing, we recommend CAR T cell dose levels be based on the number 

of viable transduced CAR T cells in the product, rather than the total cell 

number. In addition to transduction efficiency, other factors that should 

be considered in determining the dose include the total number of cells 

administered to subjects and cell viability. Sponsors can propose a flat 

dose or calculating dose based on weight or body surface area, with 

appropriate justification based on factors such as age and disease. We 

encourage sponsors to discuss their proposed dosing strategy with FDA.

b. Starting dose

The totality of available data should be considered when proposing the 

starting dose. If available, previous clinical experience with similar CAR 

T cells, even if for a different condition, may help to justify the clinical 

starting dose. However, we recommend sponsors be careful when using 

such an approach to extrapolate the starting dose as the in vivo behavior of 

CAR T cells may be different depending on the disease, antigen load, 

study population, and CAR constructs. The choice of pre-conditioning 

lymphodepletion regimen may influence CAR T cell in vivo proliferation 

and should be considered when selecting CAR T cell dose.

c. Dose escalation

Clinical development of CAR T cells has often included dose escalation in 

half-log (approximately three-fold) increments. However, the dosing 

increments used for dose escalation should consider nonclinical and any 

available clinical data regarding the risks and activity associated with the 

change in dose. The clinical protocol should provide specific criteria for 

dose escalation and de-escalation. Specifically, the clinical protocol 

should include a detailed definition of dose-limiting toxicities (DLTs) and 
Contains Nonbinding Recommendations

27

justification for exemptions of any toxicities that will not be considered as 

DLTs. Most CAR T cell toxicities appear related to the rapid release of 

large amounts of cytokines (resulting in CRS) and may be correlated to the 

activation status of the CAR T cells, which can be driven by the level of 

the tumor antigen (tumor load) in vivo. Because the tumor burden differs 

among subjects, a given dose that may be safe in one subject who has a 

low tumor burden may cause considerable toxicities at the same dose in 

another subject who has a higher tumor burden. Therefore, single-patient 

cohorts and intra-patient dose escalation are typically not suitable for FIH 

CAR T cell dose-escalation studies. Continual reassessment methods 

(CRM) may be suitable with appropriate justification.

2. Repeat dosing

CAR T cells can persist in the subject or have an extended duration of activity. In 

addition, lymphodepleting therapy before CAR T cell infusion is 

myelosuppressive, and additional lymphodepletion in the context of repeat CAR 

T cell dosing may pose life-threatening risks to subjects. Therefore, most CAR T

cell trials use a single administration or one-time dosing regimen. If proposed, 

repeat dosing should be based on a preliminary understanding of the product’s 

duration of activity and toxicity. We recommend the sponsor provide justification 

for, and strategies to mitigate risks of, the proposed dosing strategy. 

3. Staggering

When there is no previous human experience with the proposed CAR T cells or 

related product, treating several subjects simultaneously may represent an 

unreasonable risk. To address this issue, consider staggered treatment to limit the 

number of subjects who might be exposed to an unanticipated risk within a 

cohort, followed by staggering between cohorts. We recommend that the 

staggering interval, either within a cohort or between cohorts: (1) be long enough 

to monitor for acute and subacute adverse events prior to treating additional 

subjects at the same dose or prior to increasing the dose in subsequent subjects;

(2) consider the time course of acute and subacute adverse events that were 

observed in the animal studies and in previous human experience with related 

products; (3) consider the expected duration of product activity; and (4) be 

practical in the context of overall development timelines.

4. Consideration for manufacturing delay or failure

Autologous CAR T cells are manufactured separately for each subject in a trial,

and this manufacturing process may take many weeks. During this period, the 

subject might have disease progression or deteriorating condition and no longer 

meet the eligibility requirements at the time of planned lymphodepletion and 

product administration. To mitigate this risk that the subject would become 

ineligible, the enrollment criteria may need to include factors that evaluate 
Contains Nonbinding Recommendations

28

whether the subject will still be eligible for product administration when the 

manufacturing process is complete. Alternatively, the trial might include separate 

criteria (i.e., different than the study enrollment criteria) that need to be met at the 

time of lymphodepletion and product administration. 

In some situations, manufacturing failures can happen, leading to unavailability of 

products for a given subject. It is important to gain an understanding from earlyphase trials of the likelihood of manufacturing failure and any subject factors that 

may relate to such failures (e.g., subject characteristics that might predict a poor 

cell harvest). This information can facilitate design of subsequent trials by 

suggesting subject selection criteria to reduce the chance of manufacturing failure, 

or by prompting the development of a treatment protocol with a formalized 

manufacturing failure contingency plan.

To mitigate risk to subjects from production-related (i.e., manufacturing) failures, 

the protocol should be designed so that the subject is not committed to receive 

high-risk lymphodepleting regimen until it is known that the product is available.

The protocol should also clearly specify whether a new attempt for treatment will 

be made with another round of manufacturing and whether an untreated subject 

will be replaced by increasing enrollment. Failure-to-treat may be an important 

trial endpoint that is part of a feasibility evaluation, and there should be plans to 

analyze the proportion of failure-to-treat subjects to look for factors that may 

predict failure to administer the product and to evaluate the consequences to the 

subject if there is a failure-to-treat.

5. Bridging therapy

A manufacturing delay or failure may prompt the investigators to use “bridging 

therapy” in an attempt to ameliorate the underlying disease while the subject waits 

for the production of the CAR T cells. However, such bridging therapy could 

confound the interpretation of treatment effects from the subsequent CAR T cells

because it may be difficult to ascertain whether any tumor response observed in

these subjects is due to the prior bridging therapy or due to the CAR T cells or 

both. In addition, lack of bridging therapy standardization can further complicate

the interpretation of the CAR T cell clinical trial results. To help understand the

impact of any bridging therapy on the interpretation of the overall study results, 

we recommend that sponsors consider reassessing baseline disease before 

lymphodepletion and conducting separate pre-specified analyses for: (1) all 

subjects; (2) subjects who received prior bridging therapy; and (3) subjects who 

did not receive prior bridging therapy.

C. Clinical Pharmacology Considerations

Clinical pharmacology assessment for CAR T cells includes pharmacokinetic (exposure), 

pharmacodynamic (response) and immunogenicity studies. PK and PD assessments 

provide important information for determination of the safety and effectiveness of drug 
Contains Nonbinding Recommendations

29

products. Immunogenicity assessments evaluate potential risks posed by immune 

responses to CAR T cells. 

1. Pharmacokinetics 

CAR T cells are living drugs capable of proliferation after administration. 

Therefore, conventional absorption, distribution, metabolism and elimination 

(ADME) criteria cannot be applied to model the pharmacokinetics of CAR T 

cells. After administration, CAR T cells expand and persist in the human body. 

Samples, such as blood samples, should be collected with a specified schedule to 

monitor in vivo persistence and proliferation of CAR T cells. To better 

characterize the kinetic profiles of CAR T cells, additional PK samples may be 

collected from certain disease-related tissue(s). For systemic exposure, the 

sponsor should collect blood samples with sufficient sampling time points to 

derive a CAR T cell concentration-time curve. We recommend the following PK

measures pertaining to CAR T cell expansion and persistence: peak exposure 

(Cmax); time to reach peak exposure (Tmax); partial area under the curve 

(pAUC); last observed measurable concentration (Clast); and time of Clast. To 

evaluate the association between CAR T cell expansion and clinical outcomes, 

CAR T cell expansion rate can be assessed and used for correlative analysis 

between exposure and efficacy and/or safety. To evaluate factors which may 

affect CAR T cell in vivo expansion and persistence, patient-related factors,

product-related factors, and concomitant therapies should be considered. Patientrelated factors include, but are not limited to, age, sex, levels of targeted antigen 

expression, and tumor burden. Product-related factors include, but are not limited 

to, CAR T cell composition and differentiation status. 

To characterize CAR T cell in vivo kinetics, we recommend that the PK sampling 

schedule include sufficient time points especially during the expansion phase, 

which is usually around the first two weeks post-infusion. The persistence of 

CAR T cells may be monitored by measuring levels of transgene and CAR 

expression. To explore the relationship between CAR T cell exposure and 

response, we recommend sponsors perform, if possible, functional analysis

(immunophenotyping) of CAR T cells. 

2. Pharmacodynamics 

Upon binding specifically to antigen-expressing cells, CAR T cells initiate 

signaling cascades to promote T cell activation, proliferation, acquisition of 

effector functions, and production of cytokines and chemokines. These events 

lead to elimination of target cells. CAR T cell pharmacodynamic assessment 

includes monitoring changes in levels of cytokines, chemokines, effectors, blood 

immunophenotyping, and clinical endpoints (such as tumor cell killing). We 

recommend that the sponsor select pharmacodynamic biomarkers based on the

CAR T cell mechanism of action, target disease-specific attributes, and clinical 
Contains Nonbinding Recommendations

30

outcomes. The PD sampling scheme should reflect the characteristics of PD 

biomarkers and anticipated duration of response. 

To improve the CAR T cell safety and effectiveness profile, we recommend 

assessing the following exploratory correlative analyses: (1) the relationship 

between CAR T cell final product characteristics and CAR T cell 

pharmacokinetic profiles; and (2) the relationship between CAR T cell exposure 

and responses using clinical PK and PD data. 

3. Immunogenicity

An immunogenicity assessment is important due to the potential impact of 

immunogenicity on clinical outcomes. We recommend developing assays to 

detect humoral and/or cellular immune responses against the CAR T cells (CAR 

and co-expressed transgenes, if applicable) during product development. Both 

patient-related and product-related factors which may affect CAR T cell 

immunogenicity should be considered. Patient-related factors include genetics, 

age, sex, disease status, general immune status, pre-existing antibody(ies) against 

the CAR T cells, and concomitant medication. Product-related factors include:

CAR T cell origin (autologous or allogeneic); CAR molecular structure and 

posttranslational modifications; co-expressed transgenes; product impurities;

formulation excipients; and container closure materials. 

For PK, PD, and immunogenicity sample analysis, assays may be developed and 

refined throughout product development. We recommend using validated 

bioanalytical methods for clinical studies intended to provide primary evidence of 

effectiveness to support a marketing application (Ref. 46).

D. Safety Evaluation and Monitoring 

CAR T cell safety considerations include the risks associated with: (1) cell procurement;

(2) concomitant therapy (e.g., the use of immunosuppressive lymphodepleting regimen 

prior to CAR T cell administration); and (3) CAR T cells.

1. Clinical monitoring

We recommend the clinical protocol include a detailed monitoring plan that is 

adequate to protect the safety of subjects. The elements, procedures, and 

schedules of the monitoring plan should be based upon available information,

including nonclinical and prior clinical experience with the proposed product or 

related products. For a FIH product, or a product with limited prior human 

experience, to minimize the possibility that subjects are exposed to unacceptable 

toxicities, staggered enrollment should be considered (see section VI.B.3 of this 

guidance).
Contains Nonbinding Recommendations

31

A particular concern of CAR T cell toxicity is CRS (see section VI.D.2 of this 

guidance). A management plan or algorithm, based on clinical signs and 

symptoms for administering anti-cytokine therapy (e.g., tocilizumab), should be 

described. 

CAR constructs are engineered genes that are not naturally occurring and,

therefore, contain components that are not endogenous to the recipient. When 

administered, these exogenous components may elicit immune responses with the 

potential to affect CAR T cell persistence or counteract the effect (anti-tumor 

activity or toxicities) of CAR T cells. We recommend that CAR-reactive immune 

responses be monitored. For example, some CAR T cells may include murinederived sequences and thus may generate human anti-mouse antibody (HAMA). 

We encourage sponsors to describe their plan and appropriate test(s) for such 

monitoring, along with a management plan to address the results of such 

monitoring. 

2. Toxicity grading

We recommend the clinical trial protocol include a toxicity grading system to 

inform decision-making such as dose escalation and patient management. We 

recommend that sponsors use the National Cancer Institute (NCI) Common 

Terminology Criteria for Adverse Events (CTCAE) for grading toxicities, with 

specific considerations for CRS and neurotoxicity. A management algorithm for 

these toxicities should be described in detail. 

CRS and neurologic adverse reactions are toxicities associated with CAR T cells

that can be life-threatening and fatal. Thus, prompt recognition and appropriate 

management of CRS and neurologic toxicities are integral to clinical trial design. 

We recommend that sponsors consider using standardized consensus criteria14 for 

grading CRS and neurologic toxicities with justification for the grading criteria

chosen. Psychiatric toxicities may also be associated with CAR T cells and 

should be assessed, reported, and managed appropriately.

3. Dose-limiting toxicities (DLTs), stopping rules and attribution

a. DLT definition

We recommend DLTs be well defined in the clinical protocol. The

definition should include CRS toxicities. The following are examples of

treatment-emergent CAR T cell DLTs:

• Any Grade 4 or 5 CRS;

• Any Grade 3 CRS that does not resolve to ≤ Grade 2 within 7 days;

14 See, e.g., Ref. 47.
Contains Nonbinding Recommendations

32

• Any autoimmune toxicity ≥ Grade 3;

• Grade 3 and greater neurotoxicity;

• Grade 3 and greater allergic reactions related to the cell infusion; 

and

• Grade 3 and greater organ toxicity (cardiac, dermatologic, 

gastrointestinal, hepatic, pulmonary, or renal/genitourinary) not 

pre-existing and not due to the underlying malignancy and 

occurring within 30 days of cell infusion.

The DLT definition may vary depending on many factors, such as the 

underlying disease, CAR T cell characteristics, and the grading system 

used to characterize the toxicities. Any exception or exemption of 

treatment-emergent toxicities from the DLT definition should be clearly 

described and justified. In addition, the observation period for DLTs 

should be adequate to capture both acute and delayed toxicities. 

b. Attribution

It is often difficult to attribute an observed treatment-emergent toxicity to 

a specific cause during the clinical study due to confounding factors such 

as the symptoms of the underlying disease, concomitant treatment, and 

CAR T cell therapy. Therefore, we recommend DLTs be defined 

independent of attribution to CAR T cells unless a clear alternative cause 

can be described.

c. Stopping rules

Stopping rules are criteria for halting the study based on the observed 

incidence of particular adverse events. The objective of study stopping 

rules is to limit subject exposure to risk in the event that safety concerns 

arise. Well-designed stopping rules may allow sponsors to assess and 

address risks identified as the trial proceeds, and to amend the protocol to 

mitigate such risks or to assure that human subjects are not exposed to 

unreasonable and significant risk. Examples of stopping rules for CAR T 

cell clinical studies may include an increase in the number or frequency of 

expected severe adverse events, unexpected severe adverse events (e.g., > 

two (2) Grade 4 CRS for a FIH CAR T product), or any death within the 

30 days after CAR T cell administration.

E. CAR T Cell Persistence and Long Term Follow-up

We recommend the clinical protocol describe the plans to determine the duration or 

persistence of the administered CAR T cells in trial subjects. The specimens for such a 

determination may include blood, body fluids, and tissues. If an invasive procedure is

used to procure the specimen, a separate informed consent is recommended to inform the 

trial subjects of the risks of the procedure. Analytical methods for assessing CAR T cell 
Contains Nonbinding Recommendations

33

persistence should be described in detail. Such methods could include tests for the 

presence of CAR T cells, or vector, and for the activity of the CAR T cells, including 

gene expression or changes in biomarkers.

If death occurs during the trial, planning for postmortem studies to assess the cause of 

death, including CAR T cell persistence, toxicity, and activity, should be considered.

The duration of follow-up for subjects who have received CAR T cells depends on the 

underlying disease, persistence of the CAR T cells, and the CAR vector. Subjects should 

be followed for 15 years after treatment with CAR T cells containing an integrated 

transgene. FDA recommends that a plan be provided for follow-up, including funding, in 

the event the sponsor ceases to operate or decides to inactivate, transfer, or withdraw the 

IND before completion of the long term follow up. For additional information on long 

term follow-up for CAR T cells, please refer to FDA’s guidance entitled “Long Term 

Follow-Up After Administration of Human Gene Therapy Products; Guidance for 

Industry,” January 2020 (Ref. 10).

F. Allogeneic CAR T Cells 

In addition to all of the clinical considerations discussed above, there are additional 

considerations for CAR T cells derived from allogeneic sources. We recommend the 

clinical protocol describe whether there is a plan for immunological matching of the 

donor and recipient, and if so, clearly describe the methods for such matching. In 

addition, a major concern for recipients of allogenic CAR T cells is GVHD. Clinical 

monitoring should include plans to collect information regarding the symptoms and signs 

of GVHD. A grading system used to assess GVHD (Ref. 48) and a corresponding 

management algorithm should be included in the clinical protocol. Furthermore, DLT 

and study stopping rules should incorporate GVHD.15

15 FDA’s GE Guidance also addresses additional clinical considerations for allogeneic CAR T cells that incorporate 

human genome editing (Ref. 15). 
Contains Nonbinding Recommendations

34 

VII. REFERENCES 

1. Formal Meetings Between the FDA and Sponsors or Applicants; Draft Guidance for 

Industry, December 2017, https://www.fda.gov/media/109951/download.* 

2. Guidance for Industry: Q8, Q9, & Q10 Questions and Answers -- Appendix: Q&As 

from Training Sessions (Q8, Q9, & Q10 Points to Consider), July 2012, 

https://www.fda.gov/media/83904/download. 

3. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy 

Investigational New Drug Applications (INDs): Guidance for Industry, January 2020, 

https://www.fda.gov/media/113760/download. 

4. Jensen, M.C. and S.R. Riddell, Designing chimeric antigen receptors to effectively and 

safely target tumors. Curr Opin Immunol, 2015. 33: p. 9-15. 

5. June, C.H. and M. Sadelain, Chimeric Antigen Receptor Therapy. N Engl J Med, 2018. 

379(1): p. 64-73. 

6. Weinkove, R., et al., Selecting costimulatory domains for chimeric antigen receptors: 

functional and clinical considerations. Clin Transl Immunology, 2019. 8(5): p. e1049. 

7. Alabanza, L., et al., Function of Novel Anti-CD19 Chimeric Antigen Receptors with 

Human Variable Regions Is Affected by Hinge and Transmembrane Domains. Mol Ther, 

2017. 25(11): p. 2452-2465. 

8. Hudecek, M., et al., The nonsignaling extracellular spacer domain of chimeric antigen 

receptors is decisive for in vivo antitumor activity. Cancer Immunol Res, 2015. 3(2): p. 

125-135. 

9. Jonnalagadda, M., et al., Chimeric antigen receptors with mutated IgG4 Fc spacer avoid 

fc receptor binding and improve T cell persistence and antitumor efficacy. Mol Ther, 

2015. 23(4): p. 757-768. 

10. Long Term Follow-up After Administration of Human Gene Therapy Products: 

Guidance for Industry, January 2020, https://www.fda.gov/media/113768/download. 

11. Tasian, S.K., et al., Optimized depletion of chimeric antigen receptor T cells in murine 

xenograft models of human acute myeloid leukemia. Blood, 2017. 129(17): p. 2395-

2407. 

12. Budde, L.E., et al., Combining a CD20 chimeric antigen receptor and an inducible 

caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive 

immunotherapy for lymphoma. PLoS One, 2013. 8(12): p. e82742. 

13. Di Stasi, A., et al., Inducible apoptosis as a safety switch for adoptive cell therapy. N 

Engl J Med, 2011. 365(18): p. 1673-1683. 

14. Kamiya, T., et al., A novel method to generate T-cell receptor-deficient chimeric antigen 

receptor T cells. Blood Adv, 2018. 2(5): p. 517-528. 

15. Human Gene Therapy Products Incorporating Human Genome Editing: Guidance for 

Industry, January 2024, https://www.fda.gov/media/156894/download. 

16. Guidance for Industry: M4Q: The CTD – Quality, August 2001, 

https://www.fda.gov/media/71581/download. 

17. Guidance for Industry: CGMP for Phase I Investigational Drugs, July 2008, 

https://www.fda.gov/media/70975/download. 

18. Guidance for Industry: Q2B Validation of Analytical Procedures: Methodology, May 

1997, https://www.fda.gov/media/71725/download. 
Contains Nonbinding Recommendations

35 

19. Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication 

Competent Retrovirus During Product Manufacture and Patient Follow-up: Guidance for 

Industry, January 2020, https://www.fda.gov/media/113790/download. 

20. Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, 

and Cellular and Tissue-Based Products, August 2007, 

https://www.fda.gov/media/73072/download. 

21. Elavia, N., et al., Effects of starting cellular material composition on chimeric antigen 

receptor T-cell expansion and characteristics. Transfusion, 2019. 59(5): p. 1755-1764. 

22. Stroncek, D.F., et al., Elutriated lymphocytes for manufacturing chimeric antigen 

receptor T cells. J Transl Med, 2017. 15(1): p. 59. 

23. Stroncek, D.F., et al., Myeloid cells in peripheral blood mononuclear cell concentrates 

inhibit the expansion of chimeric antigen receptor T cells. Cytotherapy, 2016. 18(7): p. 

893-901. 

24. Levine, B.L., et al., Global Manufacturing of CAR T Cell Therapy. Mol Ther Methods 

Clin Dev, 2017. 4: p. 92-101. 

25. Vormittag, P., et al., A guide to manufacturing CAR T cell therapies. Curr Opin 

Biotechnol, 2018. 53: p. 164-181. 

26. Guidance for Industry: Q8(R2) Pharmaceutical Development, November 2009, 

https://www.fda.gov/media/71535/download. 

27. Federal Register: Amendments to Sterility Test Requirements for Biological Products; 

Final Rule (77 FR 26162, May 3, 2012), https://www.govinfo.gov/content/pkg/FR-2012-

05-03/pdf/2012-10649.pdf. 

28. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current 

Good Manufacturing Practice, September 2004, 

https://www.fda.gov/media/71026/download. 

29. Guidance for Industry: Analytical Procedures and Methods Validation for Drugs and 

Biologics, July 2015, https://www.fda.gov/media/87801/download. 

30. ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text and 

Methodology Q2(R1), November 2005, https://www.fda.gov/media/152208/download. 

31. Hacein-Bey-Abina, S., et al., LMO2-associated clonal T cell proliferation in two patients 

after gene therapy for SCID-X1. Science, 2003. 302(5644): p. 415-419. 

32. Heinrich, T., et al., Mature T-cell lymphomagenesis induced by retroviral insertional 

activation of Janus kinase 1. Mol Ther, 2013. 21(6): p. 1160-1168. 

33. Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products, January 

2011, https://www.fda.gov/media/79856/download. 

34. Guidance for Industry: Studying Multiple Versions of a Cellular or Gene Therapy 

Product in an Early-Phase Clinical Trial, November 2022, 

https://www.fda.gov/media/152536/download. 

35. ICH Harmonized Tripartite Guideline: Comparability of Biotechnological/Biological 

Products Subject to Changes in their Manufacturing Process: Q5E, November 2004, 

https://www.fda.gov/media/71489/download. 

36. Guidance for Industry: Q5E Comparability of Biotechnological/Biological Products 

Subject to Changes in Their Manufacturing Process, June 2005, 

https://www.fda.gov/media/71489/download. 
Contains Nonbinding Recommendations

36 

37. Comparability Protocols for Human Drugs and Biologics: Chemistry, Manufacturing, 

and Controls Information: Draft Guidance for Industry,* April 2016, 

https://www.fda.gov/media/97148/download. 

38. Contract Manufacturing Arrangements for Drugs: Quality Agreements; Guidance for 

Industry, November 2016, https://www.fda.gov/media/86193/download. 

39. Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene 

Therapy Products, November 2013, https://www.fda.gov/media/87564/download. 

40. Fujiwara, K, et al., Hinge and transmembrane domains of chimeric antigen receptor 

regulate receptor expression and signaling threshold. Cells, 2020. 9(5): p. 1182. 

41. In Vitro Companion Diagnostic Devices: Guidance for Industry and Food and Drug 

Administration Staff, August 2014, https://www.fda.gov/media/81309/download. 

42. Investigational In Vitro Diagnostics in Oncology Trials: Streamlined Submission Process 

for Study Risk Determination: Guidance for Industry, October 2019, 

https://www.fda.gov/media/112605/download. 

43. Principles for Codevelopment of an In Vitro Companion Diagnostic Device with a 

Therapeutic Product: Draft Guidance for Industry and Food and Drug Administration 

Staff, July 2016, https://www.fda.gov/media/99030/download.* 

44. Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene 

Therapy Products: Guidance for Industry, June 2017, 

https://www.fda.gov/media/106369/download. 

45. Ethical Considerations for Clinical Investigations of Medical Products Involving 

Children; Draft Guidance for Industry, Sponsors, and IRBs, September 2022, 

https://www.fda.gov/media/161740/download.* 

46. Bioanalytical Method Validation: Guidance for Industry, May 2018, 

https://www.fda.gov/media/70858/download. 

47. Lee, D.W., et al., ASTCT Consensus Grading for Cytokine Release Syndrome and 

Neurologic Toxicity Associated with Immune Effector Cells. Biol Blood Marrow 

Transplant, 2019. 25(4): p. 625-638. 

48. Harris, A.C., et al., International, Multicenter Standardization of Acute Graft-versus-Host 

Disease Clinical Data Collection: A Report from the Mount Sinai Acute GVHD 

International Consortium. Biol Blood Marrow Transplant, 2016. 22(1): p. 4-10. 

* When finalized, this guidance will represent FDA’s current thinking on this topic.