a b

scFv (FMC63)
CD19.28 B7-H3.28 B7-H3.BB
TM
100
CD19.28 VL VH CD8α H CD28 CD3ζ Isotype B7-H3.CAR

% of max
80 CD19.CAR
scFv (376.96) 60 95 93 94
TM
40
B7-H3.28 VL VH CD8α H CD28 CD3ζ 20

0
B7-H3.BB VL VH CD8α H 4-1BB CD3ζ
CAR

c 100 d
A549 H1299 H520 H460 SK-MES-1
80 100
% of CAR +

% of max
80
60 60

40
40 20
0
20 2
10
3
10
4
10
5
10
2
10 10
3
10
4 5
10 10
2 3
10
4
10
5
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10 10
5

CFSE
0 B7-H3.28 only B7-H3.28 + tumor
CD19.28 B7-H3.28 B7-H3.BB B7-H3.BB only B7-H3.BB + tumor

e LU6438B LU6438B + CD19.28 LU6438B + B7-H3.28

Day 1

Day 2

Supplementary Figure 1. CAR structure and functional characterization of CAR-Ts. (a) Schematic of the
CD19.28, B7-H3.28 and B7-H3.BB CAR vectors. (b) Representative flow plots showing CAR expression of T
cells transduced with CD19.28, B7-H3.28 or B7-H3.BB vectors. (c) Summary of CAR expression in CD19.28,
B7-H3.28 and B7-H3.BB CAR-Ts (n = 11 independent experiments using CAR-T cells generated from 11
different donors). Data are presented as mean values +/- SD.(d) CFSE dilution of CFSE-labeled B7-H3.28 and
B7-H3.BB CAR-T cells co-cultured with NSCLC cell lines for 5 days at 1 to 1 ratio analyzed by flow cytometry.
CFSE-labeled CAR-T cells alone were used as control. (e) Representative microscopy images showing the
lung cancer organoid LU6438B cocultured with CD19.28 or B7-H3.28 CAR-Ts at the CAR-T cells to organoid
cells ratio of 1 to 2 after 24h and 48h of co-culture. This experiment was repeated two times independently with
similar results. Scale bars, 300 µm. Source data for (c) are provided as a Source Data file.
a H520, i.v. CAR-Ts, i.v. Weekly
b CD19.28 B7-H3.28 B7-H3.BB
Days X 107
1.5x106 5x106 imaging 1.0

0
Day -14 Day 0 0.8

c 9 0.6

CD19.28
Total flux (photons/sec)

B7-H3.28 0.4
109.0 B7-H3.BB 14
0.2

108.5 21 Radiance
(p/sec/
cm2/sr)
Color scale
108.0 28
Min=4e5
Max=1e7

0 10 20 30 40 50 60 70 34
Days post T cell infusion
d
42
Percent tumor free survival

100 ** p=0.0046 (19 vs 28)

** p=0.0046 (19 vs BB)
80
56
60

40 67
CD19.28
20 B7-H3.28
B7-H3.BB
0
0 10 20 30 40 50 60 70
Days post T cell infusion

Supplementary Figure 2. B7-H3.CAR-Ts control B7-H3+ NSCLC in a metastatic model. (a) Schematic
representation of the metastatic NSCLC model in which NSG mice are inoculated i.v. with FFluc-H520 cells
and then treated with CAR-Ts by i.v. tail injection. Representative tumor images (BLI) (b) and kinetics (c) of
tumor growth. (d) Kaplan-Meier tumor free survival curve of mice in (a), (n = 3 mice in CD19.28, n = 5 mice in
B7-H3.28 and B7-H3.BB groups), **p = 0.0046 (B7-H3.28 vs. CD19.28), **p = 0.0046 (B7-H3.BB vs.
CD19.28), chi-square test. Source data for (c, d) are provided as a Source Data file.
 a A549, i.c. CAR-Ts, i.v. Weekly
b CD19.28 B7-H3.28
X 106
Days
5 x 104 2 x 106 imaging 1.0

0
0.8
Day -16 Day 0

c 7 0.6
Total flux (photons/sec)

109
0.4

15
0.2
108
Radiance
(p/sec/
CD19.28 22 cm2/sr)

107 B7-H3.28 Color scale

Min=4e5
Max=1e7

0 5 10 15 20 25 30 35 40 29
Days post T cell infusion
d 100
Percent survival

36
80
60
40 e
CD19.28 A549, i.c. CAR-Ts, i.v. Weekly
20 5 x 104 10 x 106 imaging
B7-H3.28
0
0 5 10 15 20 25 30 35 40 Day -17 Day 0
Days post T cell infusion

f CD19.28 B7-H3.28 g
Days
Total flux (photons/sec)

X 106
2.0 109
0

1.5 108
7
1.0
107
CD19.28
14 0.5 B7-H3.28
106
Radiance
0 5 10 15 20 25 30 35 40 45 50
(p/sec/
cm2/sr) Days post T cell infusion
21 Color scale
Min=1.3e5
Max=2e6 h
100
** p=0.0045
Percent survival

28 80
60
40
38
CD19.28
20
B7-H3.28
0
49 0 10 20 30 40 50 60
Days post T cells infusion
Supplementary Figure 3. B7-H3.CAR-Ts do not control A549 tumor growth in a brain xenograft model in NSG
mice. (a) Schematic of the A549 brain tumor model in which NSG mice are implanted i.c. with FFluc-A549 cells (5 ×
104 cells) and treated with low dose CD19.28 or B7-H3.28 CAR-T cells (2 × 106) 16 days after tumor implantation by
i.v. injection. (b and c) Representative tumor BLI images (b) and BLI kinetics (c) of the A549 tumor growth in the
model shown in (a). (d) Kaplan-Meier survival curves of mice in (b), n = 4 mice/group. (e) Schematic of the A549 brain
tumor model in which NSG mice are implanted i.c. with FFluc-A549 cells (5 × 104 cells) and treated with high dose
CD19.28 or B7-H3.28 CAR-T cells (10 × 106) 17 days after tumor implantation by i.v. injection. (f and g)
Representative tumor BLI images (f) and BLI kinetics (g) of the A549 tumor growth in the model shown in (e). (h)
Kaplan-Meier survival curves of mice in (e), **p = 0.0045, chi-square test, n = 5 mice/group. Days indicated in (b, f)
represent the days post T cell infusion. Source data for (c, d, g, h) are provided as a Source Data file.
 a NT CCR2a CCR2b b 150000 ****
***
100 ****
Isotype **

Cell numbers
Control ****
% of max

80
CCR2 100000 CCL2 (10 ng/ml)
60

40 50 81 96
50000
20

0
0
CCR2 NT CCR2a CCR2b
c 150000
NT
****
120000 CCR2b ****
*** ****
Cell numbers

90000
*
60000

30000

0
0 0.1 0.25 0.5 1 2.5 5 10 50
CCL2 concentration (ng/mL)

Supplementary Figure 4. CCR2b shows higher expression in T cells and promotes superior chemotaxis
towards CCL2 compared with CCR2a. (a) Representative flow plots showing CCR2 expression in T cells
transduced with vectors encoding human CCR2a or CCR2b. (b) Counting of T cells migrating in response to
CCL2 in transwell assays when T cells express either CCR2a or CCR2b (n = 3 independent experiments using
CAR-T cells generated from 3 different donors), **p = 0.0081, ***p = 0.0002, ****p < 0.0001, two-way ANOVA
with Sidak’s multiple comparisons test. Data are presented as mean values + SD. (c) Counting of T cells
expressing CCR2b and migrating in transwell assays in response to different concentrations of CCL2 (n = 3
independent experiments using CAR-T cells generated from 3 different donors), error bars denote SD. *p =
0.0326, ***p = 0.0001, ****p < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. Data are
presented as mean values + SD. Source data for (b, c) are provided as a Source Data file.
a b
Tcm Tscm CCR2b. CCR2b.
CD19.28 B7-H3.28 B7-H3.BB
Tem Teff CD19.28 B7-H3.28 B7-H3.BB B7-H3.28 B7-H3.BB
CCR2b.B7-H3.28 CCR2b.B7-H3.BB 5 61 8 35 10 63 7 33 6 53

CCR7
150
Cell numbers ( × 106 )

CD4
100
21 13 16 42 8 20 18 42 9 33

50 1 70 3 57 5 76 3 43 2 58

CD8
0
0 2 4 6 8 10
7 22 5 49 4 16 7 48 3 38
Days post transduction
CD45RA
c d
CD19.28 B7-H3.28 B7-H3.BB CD19.28 B7-H3.28 B7-H3.BB
CCR2b.B7-H3.28 CCR2b.B7-H3.BB CCR2b.B7-H3.28 CCR2b.B7-H3.BB
100 100
% in CD8+ T cells
% in CD4+ T cells

80 80
60 60
40 40
20 20
0 0
Tcm Tscm Teff Tem Tcm Tscm Teff Tem

e
CCR7

CAR+
FSC-W
SSC-A

SSC-A

Major cells Single cells

FSC-A FSC-A Goat-anti-mouse

-IgG-APC

CD45RA

Supplementary Figure 5. CCR2b co-expression in B7-H3.CAR-Ts does not impair cell expansion and
phenotypic profile. (a) T cell expansion in vitro of CD19.28, B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and
CCR2b.B7-H3.BB CAR-T cells. Days indicated represent the days post T cell transduction. Data are presented
as mean values + SD (n = 4 independent experiments using CAR-T cells generated from 4 different donors).
(b) Flow-cytometry plots showing the memory phenotype profile (Tcm, Tscm, Tem and Teff) in CD4+ or CD8+ T
cells in NT, and CAR-T cells. T cells analyzed on day 12 post T cell transduction. (c and d) Summary of Tcm,
Tscm, Tem and Teff cells in CD4+ or CD8+ cells. Data are presented as mean values + SD (n = 4 independent
experiments using CAR-T cells generated from 4 different donors). (e) Representative flow gating strategy for
the memory phenotype of the CAR-T cells showed in (b-d). Source data for (a, c, d) are provided as a Source
Data file.
 a H520, i.v. CAR-Ts, i.v. Weekly
b CD19.28 B7-H3.28 CCR2.B7-H3.28
1.5x106 4x106 imaging Days X 107
1.0
-1
Day -21 Day 0
0.8

c CD19.28
Total flux (photons/sec)

7 0.6
B7-H3.28
109.0 CCR2.B7-H3.28
0.4

108.5 14
0.2

108.0
21 Radiance
(p/sec/
cm2/sr)
107.5 Color scale
Min=3e5
0 5 10 15 20 25 30 35 Max=1e7
28
Days post T cell infusion

d A549, i.v. CAR-Ts i.v. Weekly 35

1x106 3x106 imaging

Day -19 Day 0

e f
CD19.28 B7-H3.28 CCR2.B7-H3.28
Days X 107 1010 CD19.28 B7-H3.28
Total flux (photons/sec)

2.0
CCR2.B7-H3.28
-1
109.5
1.5

109.0
8
1.0

108.5

14 0.5
108.0
0 5 10 15 20 25 30 35
Radiance
(p/sec/
Days post T cell infusion
cm2/sr)
21 Color scale
Min=5e5
Max=2e7

28

35

Supplementary Figure 6. CCR2b co-expression in B7-H3.CAR-Ts modestly enhances their antitumor

activity in NSCLC metastatic models. (a-c) Schematic representation of the metastatic NSCLC model in
which NSG mice are inoculated i.v. with FFluc-H520 cells and then treated with CD19.28, B7-H3.28, or
CCR2b.B7-H3.28 CAR-T cells by i.v. injection. (a) Schematic representation of the H520 metastatic model.
Representative images of tumor bioluminescence (BLI) (b) and kinetics (c) of tumor growth as assessed by BLI
measurements, data are presented as mean values + SD in (c) (n = 2 mice in CD19.28, n = 4 mice in B7-H3.28
and CCR2b.B7-H3.28). (d-f) Metastatic model of A549 cell line in NSG mice. (d) Schematic representation of
the metastatic NSCLC model in which NSG mice are inoculated i.v. with FFluc-A549 cells and then treated with
CD19.28, B7-H3.28, or CCR2b.B7-H3.28 CAR-T cells. Representative images of tumor BLI (e) and kinetics (f)
of tumor growth as assessed by BLI measurements, data are presented as mean values + SD in (f) (n = 2 mice
in CD19.28, n = 5 mice in B7-H3.28 and CCR2b.B7-H3.28). Days indicated in (b) and (e) represent the days
post T cell infusion. Source data for (c, f) are provided as a Source Data file.
 a b
H520, i.c. CAR-Ts, i.v. Weekly
4x104 10x106 imaging CD19.28 B7-H3.28 CCR2b.B7-H3.28
Days X 106
2.0
Day -12 Day 0 0

1.5
CD19.28 B7-H3.28
c 7
CCR2b.B7-H3.28
Total flux (photons/sec)

109.0 1.0

12
108.5
0.5

108.0 20
Radiance
(p/sec/
cm2/sr)
107.5 Color scale
26 Min=1.3e5
Max=2e6
107.0

0 10 20 30 40 50 60 70 80 90 100 42
Days post T cell infusion
d
100 56
Percent survival

80
* p=0.0498
60 70

40
CD19.28 84
20 B7-H3.28
CCR2b.B7-H3.28
0 98
0 10 20 30 40 50 60 70 80 90 100
Days post T cell infusion
e Blood Spleen Brain
2.0 p=0.4139 0.8 p=0.1149 6 ** p=0.0091
T cell number/half brain

5
1.5 0.6
% of T cell
% of T cell

4
(x 105)

1.0 0.4 3
2
0.5 0.2
1
0.0 0.0 0

Supplementary Figure 7. CCR2b co-expressing B7-H3.CAR-Ts show superior antitumor activity in the
H520 brain tumor model. (a) Schematic of the H520 brain tumor model in which NSG are implanted i.c. with
FFluc-H520 cells (4 × 104 cells) and treated with CD19.28, B7-H3.28 or CCR2b.B7-H3.28 CAR-T cells (10 ×
106) injected i.v. 12 days later. (b and c) Representative tumor BLI images (b) and BLI kinetics (c) of the FFluc-
H520 tumor growth in the model shown in (a). Days indicated in (b) represent the days post T cell infusion. (d)
Kaplan-Meier survival curves of mice in (b), *p = 0.0498, chi-square test. N = 2 mice in CD19.28, n = 5 mice in
B7-H3.28 and CCR2b.B7-H3.28 in (c and d). (e) In the separate experiment, the mice were euthanized 8 days
after CAR-T cells infusion, and human CD45+CD3+ T cells were quantified in blood, spleen and tumor by flow
cytometry. Bar graph summaries are shown (n = 5 mice/group). Data are presented as mean values + SD; **p
= 0.0091, unpaired t test with Welch’s correction and two-tailed p value calculation. Source data for (c, d, e) are
provided as a Source Data file.
a
T cell

CD3-APC-H7
SSC-A

FSC-W
Tumor cell

GFP
Major cells Living cells Single cell

FSC-A Zombie-dye FSC-A GFP

(AmCyan-510)
b
CD45-Buv-396
Organiod T cell

FSC-W
SSC-A

Count
cell

Major cells Living cells Single cell

FSC-A Viability-APC-eF780 FSC-A CD45-Buv-396

c
CCR2+
FSC-W
SSC-A

Count

Major cells Single cells

FSC-A FSC-A CCR2-BV421

d
CAR+
FSC-W
SSC-A

Count

Major cells Single cells

FSC-A FSC-A Goat-anti-human-

IgG-AF647

Supplementary Figure 8. Gating strategies. (a) Representative gating strategy for flow cytometry analysis of
the co-culture experiments with NSCLC tumor cell lines for Fig. 1d-e and Fig 4d-e. (b) Representative gating
strategy for flow cytometry analysis of the co-culture experiments with organoid cells for Fig. 1i-j. (c)
Representative gating strategy for flow cytometry analysis of the CCR2 expression in Fig. 3e-f and
Supplementary Fig. 4a. (d) Representative gating strategy for flow cytometry analysis of the CAR expression in
Fig. 4a-c and Supplementary Fig. 1b-c.
 Supplementary Table 1. Summary of chemokine genes expression in lung cancer
Brain Lung LUAD LUSC
Gene (MD) (MD) (TCGA) (TCGA)
CXCL2 9.877215 10.77373 8.51883 7.085441
CXCL1 9.234862 9.520319 7.979941 9.084662
CXCL16 8.893182 8.958561 11.36734 10.49259
CCL2 8.547023 9.242671 9.421721 9.112555
CCL18 8.484626 10.30898 10.26809 9.912602
CCL20 8.007272 8.134597 7.956865 7.646065
CCL5 7.90908 9.474124 9.61017 9.482056
CXCL12 7.868033 8.422825 9.194566 8.793509
CCL4 7.833945 7.914497 7.310597 7.19234
CXCL14 7.77247 8.338863 9.225043 10.30839
CCL3L1 7.747769 7.312868 4.438269 4.419468
CCL3 7.714433 7.285501 7.204345 7.043956
CXCL9 7.683731 10.49648 9.946797 9.648854
CXCL3 7.593487 7.701252 5.644422 5.277359
CXCL10 6.422961 7.148977 8.628386 8.889078
CXCL13 6.394719 8.37715 8.336519 8.616292
CXCL6 6.241834 5.684184 4.716799 6.771357
CCL13 6.07379 7.448635 7.203175 5.752633
CXCL5 5.976363 5.588311 6.583589 5.725901
CXCL11 5.793083 6.67535 6.27315 6.164005
CCL14 5.758235 7.356038 6.861608 5.487592
CCL19 5.753628 9.717836 7.893972 7.357034
CCL8 5.319558 5.131602 5.611836 5.241191
CCL22 5.254654 5.76306 7.533177 6.750719
CCL17 5.17511 5.328044 4.640333 3.373005
CCL26 4.770659 4.647026 2.411255 4.356511
CCL28 4.66718 3.648037 5.778392 5.401857
CCL7 4.256297 3.846952 3.314135 2.875969
CCL27 4.013291 3.230837 -0.74517 -0.00848
CCL15 3.639623 3.06721 2.768091 1.289302
CCL21 3.557192 8.834228 9.173221 8.777382
CCL16 3.399015 3.287316 0.281874 -0.12553
CCL23 3.353734 3.951275 3.465539 2.09642
CCL11 3.287908 5.747951 4.478769 5.385724
CCL25 3.20205 2.926472 1.094847 0.869083
CCL1 3.096825 3.31948 0.321498 -0.09343
CCL24 2.884338 2.904312 1.649996 1.58738
CXCL17 NA NA 11.6394 9.336873
CCL4L2 NA NA 6.308443 6.279797
CCL15 NA NA 3.26691 2.185574
CCL3L3 NA NA 1.770742 1.47255
Mean gene expression level (log2) of CCL and CXCL chemokine family members (41 genes) in 37
primary lung cancer (Lung-MD) paired with brain metastatic lesions (Brain-MD) from MD Anderson
Cancer Center data set, and in lung cancer adenocarcinoma (LUAD) (n = 517) and lung cancer
squamous cell carcinoma (LUSC) (n = 501) cohorts from TCGA data set. The data was sorted by the
expression level in the brain metastatic lesions from MD Anderson Cancer Center cohorts.