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-compliance-
regulatory-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 
 
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 
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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 
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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). 
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In general, FDAs guidance documents, including this guidance, do not establish legally 
enforceable responsibilities.  Instead, guidances describe the Agencys 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 FDAs 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).  FDAs 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). 
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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 
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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 FDAs guidance entitled Human Gene Therapy Products Incorporating Human Genome Editing:  
Guidance for Industry, January 2024 (GE Guidance) (Ref. 15).   
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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 FDAs website:  
https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapy-
guidances.  
 
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 
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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 FDAs 
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 stage-
appropriate 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. 
 
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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).   
 
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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 
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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 animal-
derived 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 
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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).  
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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 FDAs 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 safety-
 
 
6 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). 
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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)). 
 
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-  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. 
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Using the percentage of CAR-positive cells, the average VCN per CAR-
positive 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 
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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 products 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 
products 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 
DrugLimited 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). 
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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 FDAs current thinking on this topic. 
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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 
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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. 
 
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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 
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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).  
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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 cell-
derived 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 CD3 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. 
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  Contains Nonbinding Recommendations 
 
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 antigen-
independent 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 
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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 FDAs GE Guidance (Ref. 15) for 
additional nonclinical considerations.   
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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 early-
phase 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 cytokine-
release 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 
subjects 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 
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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 dose-
response relationship and severity of toxicities through parallel dose-
escalations 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 FDAs 
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.  
   
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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 
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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 products 
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 
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  Contains Nonbinding Recommendations 
 
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 early-
phase 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 
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  Contains Nonbinding Recommendations 
 
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.  Patient-
related 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 
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  Contains Nonbinding Recommendations 
 
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).  
 
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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 murine-
derived 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. 
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  Contains Nonbinding Recommendations 
 
  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 
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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 FDAs 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 FDAs GE Guidance also addresses additional clinical considerations for allogeneic CAR T cells that incorporate 
human genome editing (Ref. 15).   
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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. 
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  Contains Nonbinding Recommendations 
 
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. 
35 
  Contains Nonbinding Recommendations 
 
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 FDAs current thinking on this topic. 
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