]

Highlights
Mechanisms of Resistance to CAR-T cell Immunotherapy: Insights from a Math-
ematical Model
Daniela Silva Santurio, Emanuelle A. Paixão, Luciana R.C. Barros, Regina C. Almeida,
Artur C. Fassoni

• The research investigates relapse mechanisms in CAR-T cell therapy, a major

challenge that remains incompletely understood.
• Our mathematical model characterizes the influence of antigen density on CAR-
T-cancer cell interplay.
• The model accounts for multiple mechanisms of resistance, such as trogocytosis,
therapy pressure and mutations.

• Our work provide a valuable framework to investigate CAR-T cell dynamics

with potential to improve patient’s outcomes.
Mechanisms of Resistance to CAR-T cell Immunotherapy:
Insights from a Mathematical Model

Daniela Silva Santurioa,∗, Emanuelle A. Paixãob , Luciana R.C. Barrosc , Regina C.

Almeidad , Artur C. Fassonie
a Institutional Training Program, Laboratório Nacional de Computação
Cientı́fica, Petrópolis, 25651-075, Brazil
b Graduate Program, Laboratório Nacional de Computação Cientı́fica, Petrópolis, 25651-075, Brazil
c Center for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo, Hospital das

Clı́nicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-000, Brazil
d Computational Modeling Department, Laboratório Nacional de Computação

Cientı́fica, Petrópolis, 25651-075, Brazil

e Institute for Mathematics and Computer Science, Universidade Federal de

Itajubá, Itajubá, 37500-903, Brazil

Abstract
Chimeric Antigen Receptor (CAR)-T cell therapy long-term follow-up studies revealed
non-durable remissions in a significant number of patients. Some of the mechanisms
underlying these relapses include poor CAR T cell cytotoxicity or persistence, as well
as antigen loss or lineage switching in tumor cells. In order to investigate how antigen-
mediated resistance mechanisms affect therapy outcomes, we develop a mathematical
model based on a set of integral-partial differential equations. Using a continuous
variable to describe the level of antigen expression of tumor cells, we recapitulated im-
portant cellular mechanisms across patients with different therapeutic responses. Fitted
with clinical data, the model successfully captured the dynamics of tumor and CAR-T
cells for several hematological cancers. Furthermore, the role played by these mech-
anisms are explored with regard to different biological scenarios, such as pre-existing
or aquired mutations, providing a deeper understanding of key factors underlying re-
sistance to CAR-T cell immunotherapy.
Keywords: Antigen density, CAR-T therapy, Antigen-positive Relapse,
Antigen-negative Relapse, Mutation, Temporary Antigen Loss

1. Introduction

Chimeric antigen receptor (CAR)-T cell treatment is an immunotherapy in which T

lymphocytes are genetically modified to interact with tumor cells upon antigen binding,
triggering a powerful cytolytic response. In the past years, remarkable responses to the

∗ Corresponding
author
Email addresses: danielassanturio@gmail.com (Daniela Silva Santurio),
fassoni@unifei.edu.br (Artur C. Fassoni)

Preprint submitted to Applied Mathematical Modelling February 27, 2024

infusion of CAR-T cells in patients with relapsed and refractory hematological diseases
led to the FDA’s approval of multiple products targeting cancer cells expressing CD19
and BCMA antigens [1–5].
Despite promising results, follow-up data show that maintaining long-term remis-
sion is a significant challenge in CAR-T cell therapy. While complete remission can
be achieved in 70-90% of pediatric and adult ALL patients, long-term studies show
that 30-60% of patients relapsed, the majority occurring one year after treatment [6, 7].
Thus, a deeper understanding of the factors associated with primary and acquired resis-
tance to CAR-T therapy is necessary to decrease relapse rates and improve treatment
efficacy [8].
CAR-T cells’ functionality is mediated by antigen recognition. Several biologi-
cal mechanisms, such as expansion, cytotoxicity, and memory pool formation are in-
fluenced directly or indirectly by antigen receptor signaling. Thus, the antigen den-
sity presented by tumor cells is a major factor influencing CAR-T cell activity and
leads to two patterns of therapy failure: antigen-positive and antigen-negative relapses.
Antigen-positive relapses are typically associated with tumor cells expressing high lev-
els of antigen on their surfaces and are explained by limited persistence or low potency
of CAR-T cells. In antigen-negative relapses, tumor cells no longer display antigen
on their surfaces, resulting in tumors that evade CAR-mediated recognition and clear-
ance, despite CAR T-cell persistence. Different mechanisms may underlie this type of
relapse, including antigen loss due to genetic mutations [9], the existence of resistant
clones, the pre-therapy heterogeneity in the antigen density expressed by tumor cells,
resulting in the selection of resistant clones [10], lineage switch induced by immune
pressure [11], transitory antigen loss due to CAR-T cell contact, in a process called
trogocytosis [12] or due to non-mutational mechanisms, such as post-transcriptional
factors and the generation of alternative isoforms [13, 14]. Although these mecha-
nisms are known for leading to antigen-negative relapses, they are not fully elucidated
and require further exploration.
Extensive research has been conducted in an attempt to improve the efficacy of
CAR-T cells and overcome resistance mechanisms. Patients undergoing CAR-T im-
munotherapy already have intrinsic risk factors that cause defects in their T cells,
which, when combined with multiple prior treatments at high drug doses, may result in
a poor CAR product in terms of quality and quantity. Manufacturing CAR-T cells from
healthy donors (“off-the-shelf” CAR-T cell) [15], creating a product rich in a memory-
like phenotype [16] and, developing new CAR designs with combined co-stimulatory
domains [17] or “armored CAR T-cells” that constitutively secrete active cytokines
[18, 19] are all promising approaches. Further, sequential infusions, multi-antigen tar-
geting, and combining CAR-T cell immunotherapy with other therapies (allo-HSCT,
checkpoint inhibitors) are also new avenues to explore to enhance CAR-T effectiveness
[20, 21]. Despite these efforts, the clinical variability in progression-free or overall sur-
vival is controversial and numerous questions regarding CAR-T cell resistance remain
unanswered. Specially in the case of antigen loss, the underlying mechanisms vary,
posing a new set of challenges for CAR-T cell treatment [7].
As more data gathered from clinical trials became available over the years, there has
been a surge of interest in using mathematical models to describe different aspects of
CAR-T cell therapy. These models are valuable tools for analysing different outcomes

2
through in silico simulations and are among the most prominent methods used to study
biological systems. Using clinical data, Hardiansyah and Ng [22] demonstrated con-
nections between dose and disease burden by investigating the interplay of proinflam-
matory cytokines. Stein et al. [23] proposed the first mixed-effect model considering
different CAR-T cell phenotypes by investigating the impact of co-medications for
treating cytokine release syndrome after CAR-T cell expansion. In a preclinical model
for hematological cancers, Barros et al. [24] modeled the dynamics of two CAR-T cell
populations (effector and memory), highlighting the importance of the memory pool
formation for the response to therapy. Subsequently, Paixão et al. [25] extended this
model to the clinical scenario, resulting in a model that describes the patient-specific
multiphasic dynamics of CAR-T cell kinetics through the differentiation of functional,
memory, and exhausted phenotypes, as well as the dynamics of cancer cells. Singh
et al. [26] used a multiscale PK/PD model to study the relationship between CAR-
affinity, antigen abundance, tumor cell depletion, and CAR T-cell expansion in mouse
models. Other mechanisms have been explored over the past years, for example, the
interactions between CAR-T, cancer, and healthy cells to analyze efficacy and toxic-
ity [27, 28], the efficacy of patient preconditioning lymphodepletion therapy [29], and
the relation of CAR-T intracellular modulations and cellular response signaling [30].
There are also models for solid tumors [31–34] and, recently, Liu et al. proposed the
only model to date that explored the interactions of activated/non-activated CAR-T and
CD19+/CD19- tumor cells for patients with different therapy outcomes [35]. Reviews
on modeling CAR-T cell therapy are found in [36–38].
Although the existing models are able to capture CAR-T cell dynamics and pro-
vide usefeul insights, none of them focused on how antigen density affects cellular
mechanisms underlying heterogeneous responses. Here, we propose a mathematical
model to investigate how antigen-mediated resistance affects treatment efficacy. We
aim to capture different processes involved in the development of resistance to CAR-T
cell immunotherapy, including transient antigen loss of tumor cells due to treatment
pressure and permanent antigen loss due to mutations aiming to elucidate the specific
contribution of antigen density on CAR-T cell dynamics under different biological sce-
narios.

2. Materials and Methods

2.1. Mathematical model

We propose a model for the interactions between CAR-T and tumor cells, consid-
ering two phenotypes for CAR-T cells, effector (CT ) and memory (C M ) cells, as well
as two subpopulations for tumor cells, antigen-positive (T P ) and antigen-negative (T N )
cells. The schematic description of the model is shown in Figure 1a.
First, we describe the dynamics of the tumor cells. Experimental analysis by flow
cytometry often shows antigen density distributions of tumor cells as a two-component
log-normal function [39]. To address this heterogeneity in antigen expression, we fol-
low the modeling approach of [40] and use a continuous variable x ∈ [0, 1] to represent
antigen density, which is a normalization of the original antigen expression measures

3
(see Section SM-4.1). Then, the two distinct genotypes, T P (x, t) and T N (x, t), repre-
sent the antigen-positive (Ag+) and antigen-negative (Ag−) tumor cells with antigen
expression level x at time t.
While antigen-positive and antigen-negative tumor cells are constitutively distinct,
we assume that Ag+ cells may experience temporary loss of antigen upon CAR-T
cell contact [12] or due to post-transcriptional mechanisms [13, 14]. Thus, we define
a threshold of the antigen expression xmed that splits the phenotypic space into two
regions: cells with expression below this threshold of detection (0 ≤ x < xmed ) are
considered resistant to CAR-T cell therapy, while cells with expression above it (xmed ≤
x ≤ 1) are considered sensible (Figure 1b). Ag+ cells that are initially sensible to
treatment may become temporarily resistant due to phenotypic changes that reduce
their antigen expressions. Thus, the number of tumor cells that are sensible to CAR-T
cell therapy is defined as
Z 1
T S (t) = T P (x, t)dx . (1)
xmed

Following previous approaches [40, 41], we describe the dynamics of tumor cells
with the following system of integral-partial differential equations:

∂T P (x, t) ∂(v(x, t)T P (x, t)) ∂2 T P (x, t)

+ =σ + rT P (x, t)
∂t | ∂x
{z } | {z ∂x2 } | {z }
iii
i ii (2)
− θrT P (x, t) − γg(x) f (CT (t), T (t))T P (x, t) ,
| {z } | {z }
iv v

∂T N (x, t) 1 Z
= rT N (x, t) + θrW(x) T P (x, t)dx − γg(x) f (CT (t), T (t))T N (x, t) (3)
∂t | {z } 0 | {z }
vi | {z } viii
vii

where the total tumor cell population is given by

Z 1
T (t) = (T N (x, t) + T P (x, t)) dx (4)
0

and the initial distributions are given by

 2  2
T P0 − 12 x−xP
T N0 − 12 x−xN

T P (x, 0) = √ e dP
, T N (x, 0) = √ e dN
(5)
dP 2π dN 2π
where T P0 and T N0 are the initial numbers of antigen-positive and antigen-negative
tumor cells, respectively. As displayed in Figure 1b and specified in Equation (5),
the initial distributions of tumor cells are defined in terms of their homeostatic mean
antigen expression level (xN and xP ), as well as their corresponding standard deviations
(dN and dP ). Details regarding the parameters of the initial distributions can be found
in Section SM-4.1.
Equations (2) and (3) assume that tumor cells may experience two types of phe-
nomena: conservative interactions, that encompass all processes involving changes in

4
 (a) (b)

(c) (d) (e)

Figure 1: Modeling resistance mechanisms in CAR-T therapy. (a) Schematic description of the model struc-
ture. After infusion, effector CAR-T cells (CT ) expand in response to antigen contact, differentiate into
a memory phenotype, are suppressed by tumor cells, and undergo natural cell death. Memory cells (C M )
have a longer persistence but may also die and differentiate back into effector CAR-T cells upon antigen
re-exposure.Tumor cells are split into two constitutively different states: antigen-positive (Ag+, T P ) and
antigen-negative (Ag−, T N ). All tumor cells proliferate but only Ag+ cells mutate into Ag− cells and expe-
rience transient changes in antigen expression. Since CAR-T cytotoxicity requires antigen-binding, tumor
cells are killed with different rates, which depend on antigen-expression levels. (b) The initial distributions
of tumor cells along the antigen space are given by Gaussian distributions with means xN and xP , represent-
ing the homeostatic values of each population antigen-density, and dN and dP are the intrinsic variabilities
around this level (standard deviations). The threshold of antigen detection, xmed , defines two regions of
therapy responses: within the region 0 ≤ x < xmed , tumor cells are considered resistant, while within the
region xmed ≤ x ≤ 1, they are considered sensible to CAR-T cell therapy. (c) Treatment pressure induces
changes in antigen expression. This is modeled by the therapy-driven level of antigen-expression x̄(t) and its
dependence on the CAR-T cell load. During CAR-T cells expansion (↑ CAR-T cells), antigen-positive cells
lose antigen and escape immune response ( x̄(t) → xN ). As the number of CAR-T cells decreases (↓ CAR-T
cells), these cells can restore their antigen expression to its homeostatic level ( x̄(t) → xP ). (d) The mutation
kernel W(x) is defined as a Gaussian distribution with mean xN and standard deviation dN and denotes the
probability that an antigen-positive tumor cell will mutate into a antigen-negative tumor cell with antigen
expression x. (e) CAR-T cytotoxic function. The killing of tumor cells by CAR-T cells is regulated by
antigen receptor signaling and, since tumor cells display heterogeneous antigen expression, the function g(x)
represents the CAR-T cell cytotoxicity as an antigen-driven mechanism.

antigen expression while preserving the overall tumor cell population (terms (i) and
(ii)); and non-conservative interactions, which include all processes that do not pre-
serve the overall tumor cell population (terms (iii)-(viii)).
We assume that the advection of antigen-positive tumor cells along the antigen
space (term (i)) is related to the therapy-driven level of antigen expression x̄(t) that
encompasses a reversible antigen loss as a consequence of post-transcriptional mech-
anisms. Specifically, we assume that the antigen expression of Ag+ tumor cells under

5
therapeutic pressure changes according to the expression:
CT (t)
x̄(t) = xP − (xP − xN ) . (6)
kI + CT (t)
This formulation implies that, prior to the therapy, when there are no CAR-T cells in the
system (CT = 0), the therapy-driven level of antigen expression equals the homeostatic
mean expression level of Ag+ cells, xP , while once therapy is started, CAR-T cells
expand, and x̄(t) decreases progressively until reaching its minimum value, close to
the homeostatic expression level of Ag− cells, xN (Figure 1c). Finally, when CAR-
T cells enter the contraction phase, this process reverses, and x̄(t) gradually increases
until returning back to xP . The half-saturation constant kI regulates this phenotypic
transition. Higher values of kI are associated with a slower rate of change in antigen
expression, implying that tumor cells are less exposed to post-transcriptional events.
Therefore, the velocity field in Equation (2), advective term (i), describing the phe-
notypic drift along the antigen space is always directed towards the therapy-driven level
of antigen expression x̄(t) and is formally defined as

v(x, t) = −kP (x − x̄(t)) , (7)

in which the rate kP modulates the intensity of the advective flux and is named as
the phenotypic transition coefficient of antigen expression. Thus, when therapy de-
creases the therapy-driven level x̄(t), the distribution of Ag+ cells, T P (x, t), is driven
towards the region of low antigen-expression, which makes them temporarily resistant
to therapy. Eventually, without the pressure of therapy, x̄(t) increases until reaching the
homeostatic level xP and the drift velocity enables Ag+ cells to restore their antigen
expression.
The diffusive term (ii) in Equation (2) describes phenotypic instabilities in antigen
expression. Following [42], we employ a diffusion operator to simulate small pheno-
typic changes induced by epigenetic factors. For a more detailed explanation about the
conservative behavior of equation (2) and its corresponding steady-state solution, see
Section SM-4.2.
Regarding the non-conservative mechanisms in Equations (2) and (3), the follow-
ing assumptions are made through the terms: (iii) and (vi), tumor cells proliferate at a
rate r; (iv) and (vii), antigen-positive tumor cells mutate to an antigen-negative geno-
type during clonal division with rate θ; (v) and (vii), tumor cells are killed by CAR-T
cells at rate γg(x) f (CT , T ). It is important to note that proliferation occurs without
changing the antigen expression but mutations may drive antigen loss in a one-way
process. Specifically, a key assumption is that mutation-mediated genetic jumps on
antigen density are allowed from Ag+ to Ag− tumor cells but not backward. As in
[40], the parameter θ ∈ [0, 1] indicates the fraction of tumor cells that experience muta-
tion, so that (1 – θ) denotes the fraction of cells undergoing true division. The mutation
kernel W(x) is defined as a Gaussian distribution with mean xN and standard deviation
dN (Figure 1d),  2
1 x−x
− 12 d N
W(x) = √ e N . (8)
dN 2π

6
 The killing of tumor cells by CAR-T cells (terms (v) and (vii)) occurs at a rate γ,
and depends on the antigen density (as described by the function g(x)) and on the ratio
between CT (t) and T (t) (expressed by the function f (CT , T )). The antigen-dependent
cytotoxicity function g(x) and the lysis function f (CT , T ) are respectively given by:
xn
g(x) = g0 + (1 − g0 ) (9)
xmed n + xn
and
CT (t)
T (t)
f (CT , T ) = . (10)
a + CT (t)
d+
T (t)
Here, f (CT , T ) is a modified Hill function inspired by [25, 43]. For the cytotoxicity
function, we propose a smooth step function g(x) ∈ [g0 , 1] that modulates CAR-T cell
cytotoxicity as an antigen-driven mechanism. As displayed in Figure 1e, (1−g0 ) defines
the height of the step, and n controls the function sharpness. When 0 ≤ x < xmed (low
antigen expression), CAR-T cells eliminate tumor cells through antigen-independent
mechanisms at a small rate of γg0 , while for xmed ≤ x ≤ 1 (high antigen expression) the
maximum cytotoxicity rate of CAR-T cells, γ, is reached. A positive minimum fraction
g0 > 0 is taken by considering killing mechanisms like those due to the bystander effect,
CAR-T endogenous TCR , and other pro-inflammatory molecules [44].
Now, we describe the CAR-T cell population. Divided into two phenotypically
distinct subpopulations, with CT (t) representing the cytotoxic effector and C M (t) for
the memory CAR-T cells, CAR-T cell dynamics are based on our previous model [24]
and is described by:

∂CT (t)
" #
T S (t)
= κ(t) CT (t) − µCT (t) − ϵCT (t) + θ M C M (t)T S (t) − αCT (t)T (t) (11)
∂t A + T S (t) | {z } | {z } | {z } | {z }
| {z } ii iii iv v
i

and
∂C M (t)
= ϵCT (t) − θ M C M (t)T S (t) − µ M C M (t) . (12)
∂t | {z } | {z } | {z }
vi vii viii

Equation (11) assumes that effector CAR-T cells (i) undergo clonal expansion with
a time-dependent rate κ(t) after binding with sensible tumor cells (T S (t)); (ii) die natu-
rally or become exhausted at a rate µ; (iii) differentiate to a memory phenotype at a rate
ϵ; (iv) receive re-activated memory cells due to antigen recognition at a rate θ M T S (t);
and (v) are inhibited by tumor-induced immunosuppressive effects at a rate αT (t).
Based on our previous work [25], the expansion function κ(t) displays a high expan-
sion level that may be sustained for a certain period, forming a plateau, before finally
decreasing to a baseline level. This is formally described by
p1
κ(t) = rmin + , (13)
1 + (p2 t) p3

7
where rmin is the basal CAR-T cell expansion rate, p1 defines the initial expansion rate,
while p2 and p3 modulate the duration and the decay of the expansion rate, respec-
tively. These are patient-specific parameters and may describe heterogeneous patterns
associated with different CAR-T cell products.
Equation (12) describes the dynamics of memory CAR-T cells and assumes that
(vi) memory CAR-T cells are formed from the differentiation of effector CAR-T cells
at a rate ϵ, (vii) upon antigen contact, memory cells undergo a fast increase in metabolic
activity and immediately return to the effector CAR-T cell phenotype with rate θ M [45],
and (viii) die naturally at a rate µ M .
Descriptions of all model parameters and corresponding units are given in Table
1, while details regarding the model’s biological assumptions are provided in Section
SM-1.

2.2. Patient data, model fitting and numerics

We gathered CAR-T cell counts from 18 patients [10, 46–49] with various cancer
types, treated with CD28- or 4-1BB-based CD19 CAR-T cells, and fitted the model
parameters to display the data-informed therapy responses. Patients were grouped
according to their outcome, i.e., Complete Response (CR), Antigen-Positive Relapse
(Relapse+), and Antigen-Negative Relapse (Relapse−). Detailed information on clin-
ical data, patient-specific parameter estimation, and the numerical simulation of the
model are given in Sections SM-2 to SM-6.

2.3. Possible scenarios leading to resistance

To investigate the key processes involved in the emergence of relapse, we assumed
two scenarios:

• SC1 - Pre-Existing Antigen-Negative Cells: There is one Ag− cell within the initial
tumor burden (T N0 = 1 cell) and no genetic mutations (θ = 0) occur during therapy.

• SC2 - Random Mutations After CAR-T Cell Therapy: Initially, there are no Ag−
cells (T N0 = 0 cell) and the resistant population arises from genetic mutations (θ ,
0). To investigate the mutation rate (θ) variability between the cohorts, the minimum
fraction of CAR-T cytotoxicity was fixed at g0 = 0.015.

3. Results

We model the interactions among antigen-positive (T P ) and antigen-negative (T N )

tumor cells and effector (CT ) and memory (C M ) CAR-T cells. Each tumor subpopu-
lation exhibits phenotypic heterogeneity with respect to antigen expression x ∈ [0, 1],
which allows considering different resistance mechanisms such as trogocytosis, post-
transcriptional antigen loss, and mutations. Using the parameter values displayed in
Table SM-4, we present our main findings in this section.

8
 Table 1: Model parameters and functions with their units and biological meaning.

Parameter Unit Biological meaning

r day−1 Growth rate of tumor cells
θ − Mutation rate from antigen-positive tumor cells to antigen-negative tumor cells
A, a cell Half-saturation constants of CAR-T cell expansion and lysis function
d − Half-saturation constant of the cytotoxic effect on tumor cells
kP day−1 Phenotypic transition coefficient of antigen expression
σ day−1 Diffusion operator that represents the phenotypic changes induced by epigenetic factors
xP , xN − Homeostatic means of antigen expression of antigen-positive and antigen-negative tumor cells
dP , dN − Intrinsic variability of initial antigen-positive and antigen-negative tumor cells
xmed − Threshold of antigen detection
n − Hill coefficient of the antigen-mediated cytotoxicity function g(x)
kI cell Saturation constant that modulates the therapy-driven phenotypic transition of tumor cells
γ day−1 Maximum cytotoxic rate of effector CAR-T cells
g0 − Minimum fraction of CAR-T cells cytotoxicity
α (cell.day)−1 CAR-T cell inactivation/inhibition coefficient by tumor cells
µ day−1 Death rate (apoptosis and/or exhaustion) of effector CAR-T cells
rmin day−1 Minimum expansion rate by effector CAR-T cells
p1 day−1 Initial expansion rate by effector CAR-T cells
p2 day−1 Rate that regulates the duration of maximum expansion period of effector CAR-T cells
p3 - Expansion coefficient that regulates the decay of maximum expansion period of effector CAR-T cells
ϵ day−1 Conversion rate of effector CAR-T cells into memory CAR-T cells
θM (cell.day)−1 CAR-T cell conversion coefficient from memory to effector phenotype due to contact with sensible tumor cells
µM day−1 Death rate of memory CAR-T cells
Function Unit Biological meaning
κ(t) day−1 CAR-T cell expansion function
x̄(t) - Therapy-driven level of antigen expression
v(x, t) day−1 Drift velocity
W(x) - Mutation kernel
g(x) - Antigen-dependent cytotoxicity function
f (CT , T ) - Lysis function

3.1. Model assessment for patients with different therapy outcomes

We first evaluated how the proposed model describes the dynamics in patients ex-
hibiting distinct responses, namely Relapse−, Relapse+, and CR. We considered that
all 18 patients received a single- or 3-day split-dose infusion of CAR-T cells (see Sec-
tion SM-1 for details) and assumed a baseline tumor burden of T P0 = 107 cells due
to the lack of individual information. Specifically for patient G02, the initial distribu-
tion of antigen expression was available, consisting of 7.7% antigen-negative cells and
92.3% antigen-positive cells (see Section SM-2). Since the proposed scenarios pro-
duce similar outcomes, Figure 2 displays the simulations for SC1 while those for SC2
are displayed in Figure SM-4.
The model has successfully captured the experimentally observed CAR-T cell ki-
netics, in which CAR-T cells rapidly expand upon injection and reach their peak within
the first 2 weeks. Then, effector CAR-T cells rapidly decrease during the contraction
phase until the total population is predominantly formed by the long-lasting memory
cells, defining the persistence phase [25]. Most of the patients in the Relapse− cohort
have a large memory pool, a marked persistence phase, and it is possible to observe a
significant amount of effector CAR-T cells even if below the detection threshold (Fig-
ure 2, top panel). In the Relapse+ cohort, patients B10, L1, and L3, present a smaller

9
Figure 2: Model fits of tumor-CAR-T cell interactions for 18 patients divided into three cohorts: Relapse−
(top panel), Relapse+ (middle panel), and CR (bottom panel). The total CAR-T cell population includes
effector (CT ) and memory (C M ) phenotypes, while the total tumor population encompasses antigen-negative
(T N ) and antigen-positive (T P ) cells. The qPCR detection threshold of 2.5 × 106 cells was represented by
the gray area. The zoomed-in analysis of patient P02 was made from day 15 to day 45, while the remaining
CR patients were analyzed from day 0 to day 30. The simulations encompass various types of leukemia and
lymphoma, including B-ALL (B-cell acute lymphoblastic leukemia), ALL (acute lymphoblastic leukemia),
CLL (chronic lymphocytic leukemia), MCL (mantle cell lymphoma), and DLBCL (diffuse large B-cell lym-
phoma). These simulations consider SC1 (pre-existing Ag− cells, no mutations); corresponding fits for SC2
are presented in SM-4
.

pool of memory CAR-T cells while patients P12 and L2 present significant depletion
of effector CAR-T cells (Figure 2, middle panel). Patients with CR display a large ex-
pansion of CAR-T cells followed by a long-lasting persistence phase (Figure 2, bottom
panel). Furthermore, the model also captured sustained remissions in the CR cohort,
early relapses (B10, B18, and L1), and longer remissions followed by late relapses
(P12, L2, and L3). Finally, despite the pre-existence of antigen-negative tumor cells in
patient G02 and a lineage switch in patients M26 and P22, the model fully reproduced
their dynamics.

3.2. The interplay between tumor relapse and antigen expression

We now investigate the dynamics of tumor cells in response to antigen modulation.
To do so, two representative patients, P22 and L2, were selected based on their treat-
ment responses. They were chosen because they have both longer-term data (spanning
more than eight months) and clear, distinct phases of expansion, contraction, and per-
sistence of CAR-T cells. For the Relapse− patient (P22), CAR-T cells eliminated all
Ag+ cells even when antigen expression decreases (Figure 3a, t = 300). However,
once resistant cells emerge, whether due to their presence before treatment (SC1 ) or
resulting from subsequent mutations (SC2 ), the tumor manages to escape (Figure 3a,
t = 130). On the other hand, for the Relapse+ patient (L2), despite a decrease in anti-
gen expression during the first initial weeks, CAR-T cells are not able to eliminate all

10
Ag+ cells.
At the beginning of therapy, during CAR-T cell expansion, antigen-positive tu-
mor cells are sensible (T S ) and undergo depletion (Figure 3b, stage 1). As CAR-T
cells reach their peak, Ag+ tumor cells reach their lowest antigen expression level,
with a value of x̄(t) ≈ xN . At this stage, these tumor cells exhibit resistant phenotype

T P (x, t), SC1 T N (x, t), SC1 T P (x, t), SC2 T N (x, t), SC2

(a) Tumor Cells

(b) T S (t) Cells (c) x̄(t) (d) CAR-T Cytotoxicity

Figure 3: Tumor dynamics in response to antigen-modulation for the representative patients P22 (Relapse−
panels) and L2 (Relapse+ panels) in scenarios SC1 ( ) and SC2 ( ). (a) Tumor dynamics along the
antigen space. During the treatment, Ag+ tumor cells are killed by CAR-T cells and change their antigen
expression. Mechanisms like genetic mutations, the pre-existence of Ag− clones, or CAR-T cell impaired
cytotoxicity may lead to tumor progression. (b) Time evolution of tumor sensible cells (T S ) is defined in
four stages: 1) initial decay due to CAR-T cell expansion; 2) slower decay (SC1 ) and re-growth (SC2 ) due
to antigen loss; 3) secondary decay during antigen restoration; and 4) fast (SC1 ) and slow (SC2 ) re-growth
due to CAR-T cell persistence. (c) Time evolution of the therapy-driven level of antigen expression x̄(t).
Periods of the duration of the expansion, contraction, and persistence phases of CAR-T cells are indicated
by the letters E, C, and P, respectively. During the expansion phase (E) of CAR-T cells, driven by the
therapy pressure, Ag+ tumor cells lose antigen and evade the immune attack. As the number of CAR-T cells
decreases in the contraction phase (C), Ag+ tumor cells restore their homeostatic level of antigen expression.
(d) CAR-T cell cytotoxicity depends on the antigen density. Tumor cells with antigen down-regulation (Ag−)
are less likely to be killed by antigen-independent mechanisms, while tumor cells displaying high amounts
of antigen (Ag+) are more susceptible to being killed by CAR-T cells.

11
(Figure 3c). Consequently, CAR-T cell cytotoxicity is severely impaired (Figure 3d,
Ag−). Despite the presence of CAR-T cells in the system, antigen down-regulation
opens a window for tumor re-growth, and if this condition persists, an early escape
may occur (Figure 3b, stage 2). This dynamic is more pronounced for SC2 due to the
reduced cytotoxicity of CAR-T cells towards tumor cells with low antigen expression
( x̄(t) < xmed ). Specifically, patient L2 experiences faster changes in antigen expression
(Figure 3c). Furthermore, to control the selection and growth of resistant clones, sig-
nificantly higher levels of antigen-independent mechanisms (g0 ) are required, implying
that the hypothesis of pre-existing resistant cells prior to treatment is less likely. Nev-
ertheless, Ag+ tumor cells are not completely eliminated, indicating poor CAR-T cell
persistence or insufficient cytotoxicity towards tumor sensible cells (Figure 3d, Ag+).
Lastly, while CAR-T cells are in the persistence phase, there are no further changes in
antigen expression (Figure 3c), leading to two possible outcomes: (a) if a substantial
number of sensible tumor cells remain, effector CAR-T cells can be re-stimulated. If
not exhausted, they can control the emergence of a resistant population while killing
Ag+ cells; (b) if the residual number of sensible tumor cells is limited, effector CAR-T
cells are less stimulated and may be insufficient to avoid tumor escape (Figure 3b, stage
4).

3.3. Mechanisms underlying resistance to CAR-T cell therapy

To investigate the interplay between the resistance mechanisms and CAR-T cell
immunotherapy, we next analyze the influence of different tumor parameters on the
system dynamics in both SC1 and SC2 . To do so, we selected patients P22 and P12,
who respectively experienced negative and positive relapses, because they present dif-
ferent dynamics of both CAR-T cells and relapse of tumor cell populations. These
characteristics allow for a more clear examination of the influence of parameters as-
sociated with mechanisms underlying antigen loss, namely kP , kI , and dP . The first
parameter modulates the intensity of drift-velocity and phenotypic transition along the
antigen space (see Equation (7)), while the second modulates the sensibility of therapy-
driven antigen loss, in Equation (6). The third parameter represents the intratumoral
heterogeneity of antigen expression within the antigen-positive tumor cell population.
Our simulations for both patients are presented in Figure 4.

3.3.1. Phenotypic transition and epigenetic instabilities of antigen expression (kP )

We first varied parameter kP , as displayed in Figure 4a. Our simulations indicate
that changes in the coefficient of phenotypic transition kP had minimal effect on CAR-
T cell expansion and memory formation, but impacted effector CAR-T cell depletion
and re-expansion time span. As kP decreases, conservative effects weaken and the drift
velocity slows down. This results in tumor cells with a more heterogeneous phenotype
(flattened aspect of T P (x, t) distribution) and indicates that it takes longer for antigen-
positive tumor cells to transition back to the phenotypic region where they are sensible
to the therapy.
A reduced phenotypic transition coefficient (0.5kP ) for patient P22 resulted in a
larger population of sensible cells in SC1 , which enabled a greater stimulation of ef-
fector CAR-T cells, leading to better control of T N cells and delayed relapse. In SC2 ,

12
effector CAR-T cells suffer less depletion and had no effect on relapse time. For pa-
tient P12, the limited number of effector CAR-T cells or impaired cytotoxicity allowed
sensible tumor cells to evade CAR-T cell activity, especially in SC2 . In summary, our
findings suggest that a smaller value of phenotypic transition coefficient kP delays the
antigen-negative relapse and accelerates the antigen-positive relapse.

3.3.2. Therapy-driven antigen loss (kI )

Figure 4b shows that therapy-induced changes in antigen expression (kI ) had min-
imal effects on CAR-T cell dynamics, but impacted patient outcomes. In particular,
Ag+ tumor cells less susceptible to antigen down-regulation via post-transcriptional
events (1.5kI ) facilitated CAR-T cell binding due to a slower antigen loss, resulting in
a faster antigen-negative relapse for P22 in SC1 . In SC2 the variations evaluated for kI
had little effect on cell populations at the end of the simulation. A higher value of kI
slightly delayed relapse for P12 but didn’t affect initial tumor cell depletion.
Conversely, faster changes in antigen expression (0.5kI ) allowed more Ag+ tumor
cells to become temporarily resistant and quickly transition back to a sensible pheno-
type, maintaining a larger population of these T S cells. This led to a second expansion
of effector CAR-T cells and delayed antigen-negative relapse for P22. However, for
P12, it opened an escape window, resulting in a faster antigen-positive relapse. In sum-
mary, a smaller kI value leads to faster therapy-induced changes in antigen expression,
accelerating the antigen-positive relapse and delaying the antigen-negative relapse.

3.3.3. Intratumoral heterogeneity of antigen-positive tumor cells (dP )

To assess the role of the antigen intratumoral heterogeneity, we hypothesized that
antigen-positive tumor distribution would have the same homeostatic mean (xP ) but
different intrinsic variability of initial antigen expression (dP ). Figure 4c shows that a
more heterogeneous population of Ag+ cells (1.3d p ) is advantageous for controlling the
emergence of resistant clones but disadvantageous for controlling the proliferation of
antigen-positive tumor cells. Indeed, a larger number of Ag+ cells undergo the pheno-
typic transition, which leads to a greater number of effector CAR-T cells in the system
and delays the escape of tumor cells in patient P22. However, for patient P12, the
stronger depletion of effector CAR-T cells promotes a slightly faster antigen-positive
relapse. This behavior is more intense for SC2 , even improving CAR-T cell expansion
and forming the memory pool for P22. In this scenario, the low cytotoxicity g0 al-
lows more tumor cells to survive the CAR-T cell expansion phase, ultimately affecting
their depletion. In summary, a smaller value of dP , representing a more homogeneous
population of Ag+ tumor cells, accelerates the antigen-negative relapse and delays the
antigen-positive relapse.

3.4. CAR-T cell cytotoxicity and tumor evolution in relapsed patients

In this section, we investigate the impact of CAR-T cell cytotoxicity on tumor
evolution in relapsed patients. Keeping in mind that scenarios SC1 and SC2 assume
different values for θ, g0 , and T N0 , we selected patients who share the same parameter
values between scenarios, except for γ and a, which are parameters related to CAR-
T cell killing mechanisms. As a result, we conducted a comparative analysis of the

13
 Relapse− Relapse+
CAR-T Cells
Tumor Cells

(a)
CAR-T Cells
Tumor Cells

(b)
CAR-T Cells
Tumor Cells

(c)

Figure 4: Model simulations for different resistance mechanisms for patient P22 (first and second columns)
and patient P12 (third and fourth columns). After fitting the model, parameters related to (a) the phenotypic
transition (kP ), (b) the therapy-driven antigen loss (kI ), and (c) the intratumoral heterogeneity of antigen-
positive tumor cells (dP ) were varied to assess changes in the dynamics of effector ( ) and memory ( )
CAR-T cells, as well as changes in antigen-positive ( ) and antigen-negative ( ) tumor cells. The
corresponding scenarios are indicated in the CAR-T cell figures.

14
cytotoxicity function for patients P12 and L3 from the Relapse+ cohort and patients
M44 and P22 from the Relapse− cohort. Despite the fact that Ag+ tumor cells of
patients with positive relapse lose less antigen than in those with negative relapse, Ag+
tumor cells avoid CAR-T cell antitumor activity due to inhibitory signaling. Cancer
cells in patients from the Relapse− cohort, on the other hand, bypass CAR-T cell
activity by evading antigen binding.
The evolution of the total tumor cells over time is shown in the upper panel of
Figure 5, while the bottom panel displays the corresponding heatmaps of the CAR-T
cell cytotoxicity rate, γg(x) f (CT , T ), across the antigen space for both SC1 and SC2 .
Until CAR-T cells reach their peak time (C peak ), mainly Ag+ tumor cells are depleted
and antigen density is downregulated, leading to resistance. When Ag+ tumor cells
exhibit phenotype, CAR-T cell cytotoxicity is impaired, creating an opportunity for
tumor re-growth and possible escape. However, as CAR-T cell numbers decrease and
time approaches T min (time tumor has reached its minimum population) the therapeutic
pressure decreases, leading tumor cells to restore antigen presentation, which enables
even further depletion until T min , as can be observed in the heatmaps of Figure 5. Since
we set the minimum fraction of CAR-T cell cytotoxicity, g0 , at very low levels for all
patients in SC2 , this phenomenon is well marked with a less pronounced tumor cell
decay than in SC1 .
During tumor escape, the heatmaps in Figure 5 show that patients with negative
relapse experience a decrease in cytotoxicity, which is partially restored due to the
presence of some effector CAR-T cells in the system, which is clearly visible in patient
P22. On the other hand, for Relapse+ patients, there is a permanent reduction in CAR-
T cell cytotoxicity.

3.5. Mutation and selection of resistant cells

Now we investigate the fundamental differences between the proposed scenarios.
On one hand, we hypothesized that either antigen-negative tumor cells may arise due
to mutations (SC2 ) or they were already present before treatment (SC1 ). On the other
hand, in our model, these cells can be eliminated by antigen-independent mechanisms,
which is defined by the rate γg0 .
We associated the mutation rate θ with the progression-free survival (PFS) day for
relapsed patients and with the last follow-up day for CR patients. Figure 6a shows that
the mutation rate θ is negatively correlated with time in both groups. This means that
the lower the mutation rate, the longer is the response time. Overall, patients with Ag−
relapse showed much greater mutation rates than CR patients. Among the patients in
the Relapse− cohort, M19, and P22 showed a lineage switch (LS), while B10 and B18
presented early relapse, indicating a higher mutation rate compared to other patients in
the Relapse+ cohort.
The CAR-T cell cytotoxicity upon Ag− tumor cells (γg0 ) was assessed only for
SC1 , as g0 was the same for all patients in SC2 . Figure 6b shows that cytotoxicity has a
positive correlation with the PFS day in relapsed patients. The highest rates of cytotoxi-
city for the Relapse− group were seen in patients with either a significant initial burden
of resistant cells (G02) or a late relapse with lineage switch (P22). Patients with late
relapse in the Relapse+ and CR groups also showed significant antigen-independent
cytotoxicity, with a weak correlation between γg0 and the response time. This implies

15
 Relapse+ Relapse−

Figure 5: Different therapy scenarios assume different CAR-T cell cytotoxicity, resulting in different tumor
dynamics. The upper panel shows the time evolution of the total tumor cells T (t) for scenario SC1 ( )
and scenario SC2 ( ). The rapid depletion of tumor cells that occurs at the beginning of CAR-T cell
therapy (until C peak ) is hindered due to antigen loss. As the CAR-T cell population contracts and the therapy
pressure decreases, antigen expression is restored, and the remaining CAR-T cells further reduce the tumor
population (until T min ). However, if cytotoxicity is insufficient (Relapse+), antigen-positive tumor cells
escape; and if there is permanent antigen loss (Relapse−), the CAR-T cells cannot recognize their targets,
allowing the antigen-negative tumor cells to escape, although there is a high cytotoxic activity against Ag+
cells. The middle and bottom panels display the cytotoxicity heatmaps, γg(x) f (CT , T ), across the antigen
space for SC1 and SC2 , respectively. For patients with Relapse+, CAR-T cytotoxicity is severely impaired
due to the minimal number of effector CAR-T cells in the system and their weak antitumor activity, which
cannot avoid Ag+ cells escape. In patients with Relapse−, the mechanism of escape is related to the loss of
the target-antigen and not due to CAR-T cell cytotoxicity. The points C peak and T min represent the time when
the total CAR-T cell population reaches its peak and when the tumor has reached its minimum population.
All parameters used in this simulation are in Table SM-4.

that the hypothesis in SC2 , which assumes the absence of Ag− cancer cells, is more
plausible than the one in SC1 , which would require high antigen-independent CAR-T
cell cytotoxicity against these Ag− cells.

4. Discussion

In this study, we developed a mathematical model to investigate tumor response

to CAR-T cell immunotherapy. Specifically, we focused on understanding the resis-
tance mechanisms that may lead to tumor relapse. To achieve this, we considered
two subpopulations of tumor cells characterized by their antigen expression, namely
antigen-positive and antigen-negative cells, as well as two phenotypically different
populations of CAR-T cells encompassing memory and effector cells. Our model accu-
rately reproduced the dynamics of tumor and CAR-T cells across various diseases and
therapeutic responses. Although the simulations were performed for a specific target
antigen (CD19), there are no current limitations to applying the model to other anti-

16
 (a)

(b)

Figure 6: Scatter plots of the fundamental parameters underlying the scenarios proposed: SC1 (no mutation,
pre-existing Ag− tumor cells) and SC2 (onset of Ag− tumor cells from mutation after therapy start). LS
indicates Relapse− patients that undergo lineage switch. (a) Mutation rates (θ) and PFS or last follow-
up day for each patient in SC2 . There is a negative correlation between mutation rate θ and the response
duration, with Relapse− patients exhibiting greater mutation rates, and CR patients displaying the lowest
rates. Patient G02 was excluded from this analysis because the initial antigen distribution data was already
available so we did not consider the scenario where there would be mutations in addition to initial resistant
cells. (b) Antigen-independent killing mechanisms (γg0 ) and PFS or last follow-up day considering SC1 .
There is a positive correlation between cytotoxicity and response duration for relapsed patients while CR
patients display a weak correlation. PFS, progression-free survival.

gens. Of note, our model was able to capture differences in the inherent outcomes of
CD19 CAR-T cell treatments based on CD28 or 4-1BB co-stimulatory domains, which
demonstrates its potential to be used to analyze other CAR-T cell designs.
In a more general context of resistance to anti-cancer drugs, Greene et al. [40] pro-
posed a mathematical model that takes into account a continuous variable to describe
the cellular “level of resistance” for patients undergoing chemotherapy. Building on
this concept, Álvarez-Arenas et al. [41] analyzed a heterogeneous tumor population,
including both sensible and resistant cells, with phenotypic plasticity to evaluate the ef-
fects of various resistance mechanisms, including Darwinian selection, Lamarckian in-
duction, and nonlocal transfer of extracellular microvesicles on tumor evolution. How-
ever, regarding CAR-T cell immunotherapy, to the best of our knowledge, the work
of Liu et al. [35] presented the only model in the literature that considered antigen-
negative relapses. Unlike our model, it does not consider the level of antigen expression

17
of tumor cells during the dynamics. Through the incorporation of antigen density as
an independent variable, we were able to describe different biological mechanisms of
resistance, such as trogocytosis, post-transcriptional antigen downregulation and mu-
tations. By doing so, we provided insights into the role of each resistance mechanism
along the dynamics.
Most available therapy data report the total population of CAR-T cells, while the
number of tumor blasts is often expressed as a percentage of nucleated marrow cells.
However, there is a particular interest in describing antigen expression profiles, since
they are associated with escape from immunotherapy. For example, Nerreter et al. [39]
showed that ultra-low expression levels of CD19 are sufficient for inducing cytolytic
activity of CD19 CAR-T cells, but insufficient for inducing cytokine secretion and pro-
liferation. These features motivated us to establish a threshold for the antigen density
to regulate antigen-driven mechanisms. Overall, our simulations showed that patients
with positive relapse lose less antigen when compared to patients with negative re-
lapse. These findings reveal that antigen-dependent mechanisms play different roles
with respect to therapy outcomes.
Following Hamieh et al. [12] findings, our results show that sensible tumor cells
that are not killed during CAR-T cell expansion are susceptible to transient antigen loss,
through trogocytosis and therapy pressure. Additionally, the simulations show that in
patients with negative relapse, a slow antigen loss followed by a fast tumor depletion
does not necessarily result in durable responses. In this cohort, a larger number of
effector CAR-T cells are re-stimulated, leading to continuous tumor killing, which
ultimately helps in tumor control. On the other hand, in patients with positive relapse,
the same mechanism opens up an escape window, likely due to the different CAR-
T cell kinetics observed in this group. It is well-known that in this type of relapse,
CAR-T cells exhibit poor cytotoxicity and persistence, which means that even if tumor
cells are sensible to treatment, a low level of CAR-T cells is insufficient to control
tumor growth. A better understanding of the different mechanisms involved in the
dynamics can contribute to the development of combined therapies, new CAR designs
and infusion strategies.
Antigen binding plays a pivotal role in tumor clearance. Li et al. [50] showed
that upon CAR-T cell therapy the elimination of glioma cells was not immediate and
multiple CAR T-cells can interact with these tumor cells. They also showed that the
number of CAR-T cells binding to glioma cells depends on the antigen receptor den-
sity levels. Indeed, our model shows that, under antigen downregulation, tumor cells
bypass CAR-T cell binding, and tumor depletion is impaired. Our results showed that
antigen-mediated CAR-T cell cytotoxicity was effective in preventing the escape of
sensible cells in Relapse− patients, but could not control the emergence of resistant
clones. Targeting multiple antigens and increasing antigen presentation and recog-
nition are strategies to overcome this mechanism. In Relapse+ patients, cell-to-cell
interactions and other antigen-independent mechanisms slowed down the growth of
antigen-negative cells, although they were not fully eliminated. Nevertheless, in these
patients, after CAR-T cell contraction, the low levels of effector cells in the system
were insufficient to re-ignite an antitumor response. Enhancing CAR-T cell persis-
tence with a different CAR structure or re-infusing CAR-T cells may be alternatives to
surpass this type of escape.

18
 In their review, Lemoine et al. [51] reported that the percentage of relapses due
to CD19-loss varies from 7% to 25% in B-ALL and approximately one-third in DL-
BCL. In MCL, 23% of relapses were reported in the ZUMA-2 trial, with only one case
having undetectable CD19. Moreover, the authors indicate that relapses with a target-
Ag-negative clone may occur through immune editing, resulting in the selection of a
pre-existing Ag-negative subclone, or possibly through acquired loss of the target-Ag
that was initially expressed by the tumor cells. To further explore these hypotheses, we
defined two different scenarios: in scenario 1 (SC1 ), we assumed that the initial tumor
population would have at least 1 antigen-negative cell and that no additional mutations
occur; in scenario 2 (SC2 ), resistant cells arise due to mutations and CAR-T cell cy-
totoxicity against this population is minimum. We considered both scenarios for all
relapsed patients to evaluate their feasibility across cohorts. Our findings show that if
patients who later presented positive relapses had one resistant cell at the beginning
of the treatment (SC1 ), higher rates of cytotoxicity against antigen-low/-negative tu-
mor cells would be required. Therefore, it seems more likely that the tumors of these
patients underwent mutations during their evolution after treatment. For patients with
negative relapse, this question is still open. Since detecting such a small number of
antigen-negative cells is challenging, strategies like targeting a different antigen or us-
ing CAR-T cells as a bridging therapy should be considered.
The development of mathematical models for CAR-T cell immunotherapy remains
challenging since many mechanisms are still not fully understood. In particular, some
mechanisms such as trogocytosis have only been observed in mice, while data on
antigen-density profiles and tumor cell counts are often unavailable. One limitation
of our model is that it does not explicitly account for CAR-T cell exhaustion, which
might seem to affect the number of effector CAR-T cells in the system and modify
antigen modulation, especially in the context of therapy-induced antigen downregula-
tion. However, it should be noted that the model term associated with the mortality
of effector CAR-T cells encompasses both natural cell mortality and the reduction of
these cells due to their transition to the exhausted phenotype, as explicitly considered
in [25]. Most importantly, Paixão et al. [25] demonstrated that calculating the frac-
tion of non-exhausted CAR-T cells has the potential to serve as a predictive marker
for different responses, but its applicability in the case of relapse requires further in-
vestigation. Furthermore, incorporating the interplay between CAR-T cells and other
endogenous immune cells may help explain how immune mechanisms in the tumor
microenvironment influence treatment outcomes [22, 52]. In all of our simulations,
we made the assumption of an equal tumor burden for all patients due to the absence
of specific information. While this hypothesis undoubtedly affects the expansion and
cytotoxic capacity of CAR-T cells, and consequently, the parameters obtained for each
patient, it does not undermine the qualitative insights derived from this analysis. Con-
versely, it highlights the potential advantages of an open data policy for mathematical
modeling, particularly in the context of CAR-T cell immunotherapy, as pointed out in
[53]. Overall, despite these limitations, our proposed model successfully recapitulated
several resistance mechanisms, described antigen modulation dynamics, and generated
new insights into CAR-T cell immunotherapy.

19
5. Conclusions
The proposed model accurately reproduced the dynamics of tumors and CAR-T
cells in response to immunotherapy, focusing on the resistance mechanisms that lead
to relapse. By incorporating the level of antigen expression as a variable, the model
provided insights into the role of each resistance mechanism, shedding light on the
antigen-modulation dynamics. Understanding the dynamic features of the CAR-T
cell therapy, such as effector CAR-T cell persistence, antigen recognition, and cell-to-
cell interactions, may provide new information for developing combinatorial targeting
strategies, new CAR designs or CAR-T cell dose regimens. In future work, we aim to
explore these resistance mechanisms for other target antigens and to establish a larger
virtual cohort of patients that may help to improve our knowledge of heterogeneous
treatment responses.

Data Accessibility. All data used in this study can be downloaded from the cited
sources. All information, such as parameter values and code descriptions used to repli-
cate simulations and analysis are available in this work.

Authors’ Contributions. Conceptualization, D.S.S., E.A.P., L.R.C.B., R.C.A. and

A.C.F.; methodology, D.S.S., R.C.A. and A.C.F.; software, D.S.S.; writing—original
draft preparation, D.S.S.; writing—review and editing, D.S.S., E.A.P., L.R.C.B., R.C.A.
and A.C.F.. All authors have read and agreed to the published version of the manuscript.

Competing Interests. We declare we have no competing interests.

Funding. This research was funded by Conselho Nacional de Desenvolvimento

Cientı́fico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de
Nı́vel Superior (CAPES). Daniela S. Santurio was supported by a postdoctoral fel-
lowship from the Institutional Training Program (PCI) at Laboratório Nacional de
Computação Cientı́fica of the Brazilian National Council for Scientific and Techno-
logical Development (CNPq), Grant Number 300959/2022-2,301522/2023-5. Artur
Fassoni and Regina Almeida were partially supported by FAPEMIG RED-00133-21
and by CNPq 306588/2022-6, respectively.

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