Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses

Domain: neuroscience
Published: 2026-04-12
Editorial outcome: minor_revision

Abstract
Chimeric antigen receptor (CAR) T cells can achieve sustained clinical benefit in
B cell malignancies and autoimmune diseases. Despite the many potential advantages
over autologous products, allogeneic CAR T cells carry a higher risk of rejection, which
may limit persistence and therapeutic efficacy. We report the design and evaluation of
an optimized CD70 CAR that prevents rejection of allogeneic CAR T cells by targeting
activated alloreactive lymphocytes. Co-expression of this CSD70 CAR with a CD19 CAR
resulted in sustained CAR T cell persistence in the presence of alloreactive
lymphocytes and prolonged antitumor activity in a CD19 antigen escape model. In vivo,
CD19/CD70 dual CAR T cells eliminated B cel ls and CD70+ T cells derived from
patients with systemic lupus erythematosus in humanized mouse models, resulting in
reduced immunoglobulin productioLn. An allogeneic CD19/CD70 dual CAR T cell therapy
may therefore broaden clinical applicability while enabling the use of less intensive
lymphodepleting conditRioning regimens prior to CAR T cell infusion.

Introduction
Patient-derived T cells engineered to express a chimeric antigen receptor (CAR)
have been shown to be well tolerated and highly effective in the treatment of
hematologic malignancies and autoimmune disease (AID)1-2. Because autologous CAR
T cells are derived from the patient’s T cells, there are several limitations preventing
large-scale clinical application, including insufficient T cell yields, treatment delay due to
length of production, cost of production, and limited manufaScturing slots, among others3-
4. These issues can potentially be circumvented by using T cells derived from healthy
donors as the starting material for scalable manufacturing of allogeneic CAR T cells.
However, T cell alloreactivity due to human leukocyte antigen (HLA) mismatching
between allogeneic donor cells (herein referred to as the graft) and recipient (the host)
can result in rapid rejection of the Ladministered allogeneic CAR T cells by the patient’s
immune system and/or induce graft-versus-host disease (GvHD), limiting therapeutic
activity and increasing tRoxicity risks respectively5.

Alloreactivity mediated by T cell receptor (TCR) recognition of non-self-HLA
molecules can be avoided through several approaches. For example, ablating TCR
expression on allogeneic CAR T cells using gene-editing technologies can successfully
prevent GvHD4,6-8. Strategies to enhance the persistence and antitumor activity of
allogeneic CAR T cells by either preventing or delaying host versus graft (HvG)
alloreactivity are also being pursued9. A common approach to reduce host CD8+ T cell-
mediated rejection of allogeneic CAR T cells is to generate HLA class I-deficient cells
via genetic inactivation of the β2-microglobulin (B2M) gene10. Although this approach is
effective at reducing T cell-mediated allorejection, HLA-deficient cells become highly
susceptible to ‘missing self’ killing by natural killer (NK) cells11-12. Overexpression of NK
cell inhibitory ligands such as non-classical HLA molecules13 and CD4714 has been
pursued as an attempt to endow allogeneic B2M knockout (KO) cells with resistance to
both T and NK cell-mediated rejection. A limitation of this strategy, however, is that NK
cells are heterogeneous and not all NK cells express the targeted inhibitory receptors.
The balance between inhibition and activation of NK cells following allorecognition also
needs to be considered since some NK cell inhibitory and activating receptors share the
same ligand15. Successful inhibition of ‘missing self’ killinSg is likely to require extensive
engineering of allogeneic B2M KO CAR T cells for Rthe expression of multiple NK cell
inhibitory ligands, which may be constrained b y technical or manufacturing challenges
and would likely increase the risk of genoItoxicity.

A promising alternative antiL-rejection strategy that does not trigger ‘missing self’
killing is to selectively deplete alloreactive host lymphocytes. Since CD70 is transiently
upregulated on activateRd lymphocytes, we reasoned that targeting CD70 on alloreactive
host T and NK cells with an anti-CD70 CAR may be an effective approach for clinical
application. CD70 antibody blockade has been demonstrated to prevent T cell-mediated
rejection and promote allograft survival in mouse transplantation models16-17. CD70
expression in healthy tissues is largely restricted to activated lymphocytes and certain
subsets of antigen presenting cells18. CD70-targeting CAR T cells and monoclonal
antibodies have so far shown to be safe and well tolerated in patients19-21. Furthermore,
because CD70 is upregulated in numerous types of tumors and in T and B cells of
patients with AID22-23, this approach could potentially enable improved targeting of
heterogeneous tumors and elimination of autoreactive T and B cells.

To address immune-mediated rejection and antigen escape, which limit the efficacy of
allogeneic CAR T cell therapies, we developed an optimized anti-CD70 CAR designed
to selectively eliminate activated alloreactive host lymphocytes while preserving CAR T
cell function. We show that co-expression of this CD70 CAR with a CD19 CAR
enhances persistence of allogeneic CAR T cells, improves tumor control in models of
antigen heterogeneity, and reduces pathogenic antibody production in autoimmune
disease models. These findings establish CD70 targeting as a dual-purpose strategy
that simultaneously mitigates allorejection and broadensS antigen coverage. More
broadly, this approach provides a modular framewoRrk for engineering next-generation
allogeneic CAR T cells with improved durability and efficacy. By reducing dependence
on intensive lymphodepletion regimens aInd enabling activity across malignant and
autoimmune indications, this strategy expands the therapeutic potential of off-the-shelf
cellular immunotherapies.

Results
CD70 CAR T cells eliminate CD70+ lymphocytes and survive in an in vitro
rejection model
CD70 expression is rapidly induced on T cells upon activation24 and serves as a
marker of alloreactive T cells in allogeneic hematopoietic stem cell transplantation
recipients25. To test if CD70 is upregulated on alloreactive T cells in response to
alloantigens in vitro, we co-cultured peripheral blood mononuclear cells (PBMCs) with
allogeneic T cells and assessed the expression of activation-induced markers. CD70
was upregulated more frequently and remained on the surface of alloreactive T cells
longer than the activation-induced markers 4-1BB, CD25, and CD69 (Supplementary
Fig. 1), confirming CD70 as an appealing target of activated alloreactive T cells.
Similarly, CD70 was upregulated in T cells from systemic lupus erythematosus (SLE)
donors following stimulation with PMA and ionomycin (Supplementary Fig. 2),
suggesting that activated CD70+ T cells may also be targeted in patients with
autoimmune disease. We therefore hypothesized that allogeneic CD70 CAR T cells
could eliminate activated T cells and thus avoid alloreactive T cell-mediated rejection.

To test this, allogeneic CD70 CAR T cells were generateSd with a previously-
characterized anti-CD70 single-chain variable fragmRent (scFv)26 and used in an in vitro
model of CAR T cell rejection. PBMCs, represe nting patient ‘host’ cells, were cultured
for 7 days with unedited HLA-mismatchedI ‘graft’ T cells derived from the same healthy
donor used for allogeneic CAR T cell production. The resulting primed alloreactive host
T cells were isolated to serve as a highly reactive cell population able to quickly
recognize and reject allogeneic CAR T cells. Allogeneic CD70 CAR T cells were
produced with knockout (KO) at the TCRα constant gene (TRAC) locus to prevent graft-
versus-host alloreactivity26-27. CD70 CAR-expressing or non-transduced (NTD) control T
cells were then co-cultured with the primed host T cells (Fig. 1a). In this model,
expressing a CD70 CAR on graft T cells significantly reduced the percentage (Fig. 1b-c)
and absolute numbers of CD70+ alloreactive host T cells (Fig. 1d). Accordingly, total
host T cell counts were also reduced (Supplementary Fig. 3). Expression of the CD70
CAR significantly enhanced the survival of graft T cells, providing nearly complete
protection against rejection (Fig. 1e). Conversely, the alloreactive host T cells efficiently
rejected the non-transduced graft T cells.

To further evaluate the anti-rejection activity of the CD70 CAR against a more
diverse array of alloreactive host cells, PBMC MLR assays were performed. This assay
allows the potential for both NK and T cell alloreactivity without ‘pre-priming’ the host
cells (Fig. 1f). In this model, CD70+ host T and NK cells were eliminated when co-
cultured with CD70 CAR T cells (Fig. 1g-h). Only graft T cells expressing the CD70 CAR
survived and were protected against allorejection by host T and NK cells (Fig. 1i).
Notably, CD70neg host T and NK cells were not negatively affected by the CD70 CAR
(Fig. 1j). These data suggest that CD70 CAR T cells are Sprotected from allorejection
due to the ability to selectively eliminate alloreactivRe CD70+ host lymphocytes.

Optimization of CD70 CAR expressio n and anti-rejection function
We previously evaluatedC the activity and specificity of this CD70 CAR in renal cell
carcinoma (RCC) cell linesT and patient-derived xenograft mouse models, tissue cross-
reactivity studies, and a Phase 1 clinical trial21,28. Importantly, allogeneic T cells
expressing this CD70 CAR are fratricide-resistant due to cis-masking of CD70 and can
be manufactured in large scale without the need to knock out CD7028. However, the
CD70 CAR used in the Phase 1 TRAVERSE clinical study in clear cell RCC (ccRCC)
contains a rituximab recognition domain, which may preclude its use in patients that
have been exposed to rituximab, including patients with non-Hodgkin lymphoma or
autoimmune diseases. Given the unique properties of this CD70 CAR with regards to
manufacturability, safety, specificity, and antitumor activity, we aimed to screen and
identify variants with optimal anti-rejection activity that lack the rituximab recognition
domain. To do so we constructed a series of CD70 CAR variants (Fig. 2a), which
included CARs with a 4-1BB costimulatory domain (bbz) or without (z). In addition, we
tested variants with different transmembrane domains and linker lengths, which were
previously shown to influence CAR activity28. Only T cells expressing the Qz and z
CD70 CAR variants had significantly higher levels of transduction (Fig. 2b) and CAR
expression (Fig. 2c) compared to T cells expressing the original CAR. In addition,
CD70Qz and CD70z CAR T cells exhibited reduced or comparable activation and
differentiation compared to cells expressing variants with a costimulatory domain (Fig.
2d-e and Supplementary Fig. 4).

To evaluate anti-rejection activity, alloreactive T cell and PBMC MLR assays
were performed with CD70Qz and CD70z CAR T cells. The original CD70 CAR and the
CD70Qbbz CAR variant were also included for comparison. CD70z CAR T cells showed
significant survival in alloreactive TL cell MLR assays compared to non-transduced graft
T cells (Fig. 2f). CD70Qz also showed improved survival compared to non-transduced
graft T cells, yet the diffRerences were not statistically significant. In PBMC MLR assays,
CD70Qz and CD70z CAR T cells showed enhanced survival and suppressed host T
and NK cell expansion (Fig. 2g). Overall, T cells expressing the CD70z CAR, which
lacks the rituximab recognition domain, demonstrated survival in allorejection models
that was comparable to T cells expressing the original CD70 CAR (QR3bbz), while also
showing higher CAR expression and a favorable phenotype. Based on these findings,
we selected the CD70z CAR variant for further characterization.

Anti-rejection and anti-CD19 activity are maintained in allogeneic CD19 CAR T
cells co-expressing the CD70z CAR
To exploit the benefits of the CD70z CAR in mediating survival of allogeneic CAR
T cells, the combination of an anti-rejection CD70z CAR with a CD19 CAR was
examined. A bicistronic construct encoding a CD19/CD70 dual CAR was therefore
designed for site-specific integration (SSI) at the TRAC locus to model an ‘off-the-shelf’
allogeneic CAR T cell product. Integration of the CD19/CD70 dual CAR transgene into
the TRAC locus resulted in a high percentage and yield of CD19 CAR/CD70 CAR
double positive cells (Supplementary Fig. 5a-e). No significant differences were
observed in CD19 CAR cell surface expression and bindSing of soluble CD19 in cells
expressing the dual CAR and single CD19 CAR (SRupplementary Fig. 5f-g). It has been
previously shown that expressing two separate CARs can result in competition for
intracellular signaling molecules29. To invIestigate this, we analyzed phosphorylation of
p38 MAPK following exposure of CAR T cells to target cells expressing CD19 but not
CD70 or after stimulation of CAR T cells with PMA and ionomycin (Supplementary Fig.
5h-j). While the average frequency of p-p38+CAR+ T cells was higher for CD19/CD70
CAR T cells following short exposure to CD19-expressing cells, the difference between
the CD19 and CD19/CD70 CAR T cells did not achieve statistical significance. These
data suggest that co-expression of the CD70z CAR does not have negative effects on
CD19 CAR expression, CD19 antigen binding, or downstream signaling.

To rule out any negative effects of the CD70z CAR on CD19 CAR function, short-
term killing assays were performed at different effector to target ratios (E:T) using
CD19+ Raji lymphoma cells. Since Raji cells express high levels of CD70, CD70KO Raji
cells were also tested. The CD19/CD70 CAR T cells eliminated both CD70+ and
CD70KO Raji tumor cell targets and showed similar cytotoxicity compared to CD19 CAR
T cells (Fig. 3a). Furthermore, there were no significant differences in effector cytokine
production from CD19/CD70 CAR T cells and CD19 CAR T cells following exposure to
CD19+ target cells (Supplementary Fig. 6a). Like CD19 CAR T cells, CD19/CD70 CAR
T cells rapidly eliminated CD19+ B cells from both healthy and SLE donor PBMCs (Fig.
3b and Supplementary Fig. 6b).

The possibility that the CD70z CAR may affect long-term cytotoxic activity
against CD19-expressing target cells was also investigatedS using a serial restimulation
assay. CAR T cells were co-cultured with Raji tumor cell targets and subsequently
exposed to fresh target cells every 2-3 days until cytotoxic activity was no longer
detectable. CD70KO and CD19KO Raji tumor ce ll targets were used to assess CD19 CAR
and CD70 CAR function in isolation, respectively. Both CD19 CAR T cells and
CD19/CD70 CAR T cells persisted and maintained CD19 CAR-mediated cytotoxicity
against CD70KO Raji tumor cells for up to 9 rounds of stimulation (Supplementary Fig.
6c), indicating that co-eRxpression of the CD70z CAR does not negatively impact short-
term and long-term anti-CD19 effector function. CD19/CD70 CAR T cells also
maintained CD70 CAR-mediated cytotoxicity against CD19KO Raji tumor cells for up to
11 rounds of stimulation.

We also evaluated the cytotoxic activity of CD19/CD70 CAR T cells in an in vivo
orthotopic Raji lymphoma model. In mice bearing CD70KO Raji tumors, CD19/CD70
CAR T cells exhibited a slight but non-significant reduction in efficacy with only three of
ten mice showing tumor regrowth at approximately day 20 post CAR T cell treatment
(Fig. 3c and Supplementary Fig. 7a). However, CD19/CD70 CAR T cells showed
significantly higher peripheral expansion compared to CD19 CAR T cells (Fig. 3d).
Conversely, in mice bearing the parental Raji tumors, which express both CD19 and
CD70, CD19/CD70 CAR T cells exhibited a slight but non-significant improvement in
efficacy (Fig. 3c and Supplementary Fig. 7b). Higher peripheral expansion of
CD19/CD70 CAR T cells was also observed in mice bearing the parental Raji tumors
(Fig. 3d).

Because higher peripheral expansion of the CD19/CD70 CAR T cells was not
necessarily associated with higher efficacy in an in vivo modSel where tumor cells
express a single antigen, we hypothesized that heterodimerization of the CD19 CAR
and the CD70z CAR via the hinge domain could result in excessive stimulation and
enhanced proliferation without an increase in c ytotoxic activity. Co-expression of CARs
that harbor CD8α hinge domains for dual antigen targeting can result in CAR
heterodimerization and activation Lin trans following binding to a single antigen30-31. We
thus generated a CD19/CD70 dual CAR construct in which the CD19 CAR and the
CD70z CAR harbor a CRD8α and a CD28 hinge domain, respectively, to prevent the two
CARs from forming heterodimers.

CD19/CD70 CAR T cells expressing a CD70z CAR with a CD28 hinge domain
(herein referred to as CD8/CD28-HTM) showed levels of CAR expression and CAR T
cell yields that were comparable to T cells expressing the original construct design
(CD8/CD8-HTM), which contains only CD8α hinges (Supplementary Fig. 8a-e). To
evaluate the efficacy of CD8/CD28-HTM CAR T cells, we performed in vitro and in vivo
experiments using tumor target cells that only express the CD19 antigen. CD8/CD28-
HTM and CD8/CD8-HTM CAR T cells exhibited comparable efficacy in vitro and in vivo
(Supplementary Fig. 8f-g). In vivo, CD8/CD28-HTM CAR T cells showed a slight
reduction in efficacy compared to CD8/CD8-HTM CAR T cells for two of the three
donors tested, although these differences did not reach statistical significance.
Interestingly, peripheral expansion of CD8/CD28-HTM CAR T cells was significantly
reduced compared to CD8/CD8-HTM CAR T cells (Supplementary Fig. 8h), suggesting
that heterodimerization of the CD19 and CD70z CARs may enhance proliferation when
a single antigen is engaged. We also assessed CD70-mediated CAR T cell expansion
in PBMC MLR assays. To achieve this, host CD19+ B cells were depleted prior to co-
culture of CAR T cells and host PBMCs. Both CD8/CD28S-HTM and CD8/CD8-HTM
CAR T cells eliminated CD70+ host T cells and expRanded to similar levels
(Supplementary Fig. 8i), although CD8/CD8-HTM CAR T cells showed higher
expansion at earlier timepoints. Similar cyItotoxicity and expansion were seen when both
CD19 and CD70 antigens were present in in vitro assays (Supplementary Fig. 8f and
8j), once again with CD8/CD8-HTM CAR T cells showing higher expansion at earlier
timepoints (Supplementary Fig. 8j).

Lastly, alloreactive T cell MLR assays were performed to confirm the anti-
rejection activity of the CD70z CAR. CD19/CD70 CAR T cells significantly reduced the
number of CD70+ host T cells from healthy and SLE donors and were able to persist,
whereas CD19 CAR T cells lacking the anti-rejection CD70z CAR were eliminated by
the host T cells and were unable to suppress expansion of CD70+ host T cells (Fig. 3e
and Supplementary Fig. 6d). Taken together, these data show that the CD70z CAR and
the CD19 CAR maintain independent functions when co-expressed on allogeneic T
cells.

Dual targeting of antigenically heterogeneous tumors with CD19 CAR T cells co-
expressing an anti-rejection CD70z CAR limits antigen escape
CD70 is expressed in multiple hematologic malignancies including LBCL32 and is
emerging as a validated target for immunotherapies32-35. Therefore, we hypothesized
that the CD70z CAR could enhance cytotoxicity against a heterogeneous population of
Raji cells when co-expressed with a CD19 CAR. To test this, we used a mixed
lymphoma model to simulate tumor antigen heterogeneity. CSD19KO and CD70KO Raji
cells were mixed at a 1:1 ratio prior to co-culturing with CAR T cells (Fig. 4a). In this
model, CD19 CAR T cells were unable to eliminate all the Raji tumor cells and CD19neg
Raji cells expanded (Fig. 4b), whereas CD19/CD70 CAR T cells effectively eliminated
all the Raji cells. These results were also observed in an antigenically heterogeneous
Toledo lymphoma model (SupplemLentary Fig. 9a), demonstrating enhanced tumor
control of CD19/CD70 CAR T cells against multiple cancer cell lines in vitro.

To evaluate the CD19/CD70 CAR T cell efficacy against antigenically
heterogeneous tumors in vivo, NSG mice were implanted with a 1:1 mixture of CD19KO
and CD70KO Raji cells, treated with either CD19/CD70 or CD19 CAR T cells, and
monitored for tumor growth (Fig. 4c). CD19/CD70 CAR T cells potently eradicated
heterogeneous Raji tumors and expanded in peripheral blood, resulting in mice that
remained in remission up to the end of the study (Fig. 4d-f, and Supplementary Fig. 9b).
CD19 CAR T cells also expanded in peripheral blood, although significantly less than
CD19/CD70 CAR T cells. In contrast to CD19/CD70 CAR T cells, CD19 CAR T cells
failed to control tumor growth. These data demonstrate the enhanced efficacy of
bispecific CAR T cells and the dual-purpose function of the CD70z CAR in CD19+CD70+
malignancies.

CD19/CD70 CAR T cells eliminate B cells in CD34+ humanized mice
Since CD19 is a promising target for allogeneic CAR T cell therapy against B cell
mediated AID36, we evaluated the ability of CD19/CD70 CAR T cells to eliminate
primary human B cells in a hematopoietic stem cell (HSC) humanized mouse model.
CD34+ HSCs were engrafted into NSG mice (Hu-CD34 mice), resulting in multi-lineage
reconstitution of human immune cell populationsP. Each mouse was given one dose of
6 × 106 CAR T cells or 6 × 106 non-transduced T cells (Fig. 5a). Engrafted host B and T
cells and CAR T cells were monitoreEd in peripheral blood by flow cytometry analysis.
Host B cells were completely dCepleted by day 7 post CAR T cell injection but started to
recover and reconstitute bTeginning at day 20 post CAR T cell injection (Fig. 5b). No
significant effects were observed on the host T cell population in this model (Fig. 5b)
and CD70 expression was not detected (Supplementary Fig. 10a), which is likely
attributed to the lack of thymic education and proper T cell development in Hu-CD34
mice37. CD19/CD70 CAR T cells were detectable in peripheral blood with peak
expansion occurring on day 7 post CAR T cell injection, which was followed by
contraction of the CAR T cells (Fig. 5c). CD19/CD70 CAR T cells were nearly
undetectable in peripheral blood by 30 days post CAR T cell injection. These data
suggest that the administration of CD19/CD70 CAR T cells into mice reconstituted with
a human immune system result in deep B cell depletion without prolonged B cell
aplasia. Additionally, CD19/CD70 CAR T cells engrafted, expanded, and transiently
depleted B cells in Hu-CD34 NSG mice without the need for prior lymphodepletion or
pre-conditioning of the mice.

CD19/CD70 CAR T cells deplete B cells and reduce antibody levels in mice
humanized with PBMCs from normal and SLE donors
We sought to assess the efficacy of CD19/CD70 CAR T cells in mice engrafted
with human PBMCs. NSG mice, aged 7 to 8 weeks, were pre-conditioned with 1 Gy
irradiation and 2 x 107 PBMCs isolated from healthy human donors were adoptively
transferred by intravenous (i.v.) injection (Fig. 6aP). In this model, adoptively transferred
host B cells successfully engrafted (Fig. 6b-c) and rapidly transitioned from a
predominantly naïve B cell phenotypEe to a more differentiated phenotype in the absence
of CAR T cells (Supplementary Fig. 10b and 10c). Furthermore, host B cells produced
detectable levels of human immunoglobulin G (IgG) and M (IgM) in the serum of mice
(Fig. 6d), allowing us to determine the effects of CAR T cell-mediated B cell depletion
on immunoglobulin production. Three days after adoptive transfer of PBMCs, mice were
randomized and received a single dose of 1 × 106 CAR T cells, 4 × 106 CAR T cells, or
were left untreated. CD19/CD70 CAR T cells effectively depleted host B cells at both
dose levels, resulting in a significant reduction in human IgG and IgM production (Fig.
6b-d). CD19/CD70 CAR T cells expanded in a dose-dependent manner in both the
spleen and peripheral blood following target exposure (Fig. 6e-f). A minor population of
CD70low T cells was observed in the spleen of untreated control mice on day 10 but was
absent in mice receiving CD19/CD70 CAR T cells (Fig. 6g-h).

Next, PBMCs from SLE donors were employed in this model to test the ability of
CD19/CD70 CAR T cells and CD19 CAR T cells to eliminate autoreactive B cells and
abrogate autoantibody production. NSG mice were engrafted with PBMCs isolated from
SLE donors and received a single dose of 5× 106 CD19/CD70 CAR T cells, CD19 CAR
T cells, or non-transduced control T cells (Fig. 7a). As expected, CD19/CD70 and CD19
CAR T cells depleted host B cells and proliferated (Fig. 7b-d). However, only treatment
with CD19/CD70 CAR T cells resulted in significantly reduced host T cell counts (Fig.
7c). CD19/CD70 CAR T cells remained detectable in the spleen on day 14 post CAR T
cell injection, whereas CD19 CAR T cells were not detectable (Fig. 7b-c), suggesting
that they were likely rejected by host T cells after CAR T cell activation and expansion.
Although a reduction in IgG levels was obIserved in mice receiving CD19 CAR T cells on
day 7 post-CAR T cell injection (SLE D2), CD19 CAR T cells did not significantly reduce
the levels of α-dsDNA autoantibodies, IgG, and IgM when compared to untreated mice
on day 14 post CAR T cell injection (Fig. 7e and Supplementary Fig. 11a). For one of
the two SLE host donors tested (SLE D2), two mice receiving CD19 CAR T cells
developed GvHD and were euthanized before day 14 post CAR T cell injection. In
contrast, CD19/CD70 CAR T cells significantly reduced the production of human
immunoglobulins (Supplementary Fig. 11a) and α-dsDNA autoantibodies (SLE D1, Fig.
7e).

To further evaluate the benefits of the CD70z CAR to prevent rejection and to
assess the potential of the CD70z CAR to enable CAR T cell persistence in the absence
of prior lymphodepletion, we repeated the above experiment and included an additional
group to model lymphodepletion. Bendamustine and fludarabine/cyclophosphamide-
based lymphodepletion regimens prior to CAR T cell therapy can result in up to a 100-
fold decrease in lymphocyte counts38. We therefore adoptively transferred 100-fold less
host PBMCs into NSG mice to model a lymphodepleted host. In the lymphodepletion
(LD) group, both CD19 CAR T cells and CD19/CD70 CAR T cells could be detected in
the spleen on day 14 post CAR T cell injection (Supplementary Fig. 11b-c). In the
absence of lymphodepletion (no LD group), CD19/CD70 CAR T cells were readily
detected on day 14 post CAR T cell injection whereas CD19 CAR T cells were sparsely
present (Supplementary Fig. 11b-c), supporting the notioSn that CD19 CAR T cells are
rejected by the host PBMCs whereas the CD70z CRAR enables allogeneic CAR T cells
to persist. Taken together, these findings highl ight the ability of the CD70z CAR to
significantly enhance CAR T cell persisteInce in allogeneic hosts and support the
therapeutic potential of allogeneic CD19/CD70 CAR T cells for the treatment of
autoimmune diseases driven by autoreactive B and T cells.

Methods
Ethics statement
Leukopaks (70500) from deidentified healthy donors as well as PBMCs (70054) from
SLE donors were collected by the StemCell Technologies under IRB/REC-approved
programs (WCG and Alpha IRB) with written consent, and samples were
pseudonymized such that personal identifiers cannot be accessed. All of the mice in this
study were treated following a protocol (ALLO-001-2025) approved by the Allogene
Therapeutics Institutional Animal Care and Use Committee (IACUC).

Construction of lentiviral and adeno-associated viErus vectors encoding anti-CD70
and anti-CD19 CARs
The pLVX (Takara Bio, 632164) lentiviral vector (LVV) was employed for cloning and
expression of the CD70 CAR or CD7E0 CAR variants. An adeno-associated virus (AAV)
plasmid (Cell biolabs, Inc., VPKC-411) containing two inverted terminal repeats was
modified to incorporate two homology arms, each 300-400 bp in length corresponding to
the genomic integratAion site at the TRAC locus, which flank the nuclease target site.
The cDNA sequences corresponding to the CD19 CAR or CD19/CD70 dual CAR were
cloned downstream of the human PGK-1 promoter and are followed by the bovine
growth hormone polyA signal. All CD70 CAR constructs contained an scFv specific for
CD7028 followed by an extracellular spacer or ‘hinge’ (H) and transmembrane (TM)
domain derived from either human CD8α or human CD28, and the intracellular domains
of 4-1BB (bb) and CD3ζ (z), or the CD3ζ domain only for constructs without a
costimulatory domain. Some variants contained short mimotopes derived from either
CD20 (R mimotope) and/or CD34 (Q mimotope)28,53 that were inserted in the
extracellular domain of the CD70 CAR to increase the length of the spacer. The CD19
CAR contained the FMC63 scFv, CD8α hinge and transmembrane domain, and
cytoplasmic domains derived from 4-1BB and CD3ζ.

Cell and viruses
T cells were isolated from PBMCs using the human T Cell isolation kit
(STEMCELL Technologies, 17951). To generate Raji (ATCC, CCL-86) and Toledo
(ATCC, CRL-2631) knockout cell lines, Cas9 ribonucleoproteins (RNPs) were prepared
by incubating 60 ng of Cas9 (IDT, 1081059) with 35 pmol sgRNA (Synthego) in Neon
electroporation buffer (Invitrogen, MPK1096E). LPuciferase (Luc)-Green Fluorescent
Protein (GFP)-expressing Raji cells, generated by transduction with an LVV encoding
firefly luciferase, Tag GFP 2, and the Blasticidin resistance gene (AMSbio, LVP323-
PBS), were mixed with Cas9 RCNPs and electroporated with a Neon transfection kit
(Invitrogen, MPK1096) andT device (Invitrogen, MPK5000). Transfected cells were
expanded in RPMI 1640 medium (Gibco, 72400-047) containing 10% FBS (GE
Healthcare, SH300.70.03) and KO cells were isolated by FACS. Cell sorting was
performed on a BD FACS Aria Fusion sorter. To produce LVV, HEK293T cells (ATCC,
CRL-3216) were transfected with lentiviral transfer vector pLVX, psPAX2 (Elim Pharma,
20838-1), and pMD.2G (Elim Pharma, 20838-2) using Lipofectamine 2000 (Invitrogen,
11668500). 24 hours after transfection, the medium was replaced with X-Vivo 15
medium (Lonza, BP02-054Q), supplemented with 10% FBS. Supernatant was then
harvested and passed through a 0.45 µM filter (Thermo Fisher Scientific, 723-9945) 24
hours later. All AAV vectors used in this study were produced using a triple transfection
with AAV-MAX system according to the manufacturer's protocol (Thermo Fisher
Scientific, A50516). Briefly, the pRC6, pHelper, and transgene plasmids were mixed at
the molar ratio of 1:1:1 for gene transfection. After 72 hours, cells were lysed with the
AAV-MAX lysis buffer (Thermo Fisher Scientific, A50520) for 2 hours and cell debris
were removed by centrifugation and filtration with 0.2 µM filter (Nalgene, 09-741-88).
The clarified lysate was loaded on a HiTrap AVB column (Cytiva, 17372211) and
column-bounded AAV particles were eluted with 0.1 M NaOAc, 0.5 M NaCl, pH 3.0. The
elution solution was immediately neutralized with 1M Tris-HCl, pH 8.8, further
concentrated, and exchanged to PBS (Corning, 21-040-CSM). The titers of the
concentrated AAVs were determined by qPCR (TaRkara, 6233).

CAR T cell production
The CD70 CAR T cells used in figures 1 and 2 were produced with LVV as previously
described28. To generate CTAR T cells via site-specific integration, human T cells were
first activated with T cell TransAct reagent (Miltenyi Biotec, 130-111-160) as
recommended by the manufacturer protocol and cultured in X-Vivo15 medium
supplemented with 5% human AB serum (Gemini Bio-Products, 535-100-HI GI) and 100
IU/mL IL-2 (Miltenyi Biotech, 130-097-748). Three days post-activation, cells were
washed in PBS and electroporated using the AMAXA 4D nucleofector electroporation
apparatus with 15 µM of RNP, TRAC crRNA54 combined with ABR-001 nuclease, for
every 3-12 x 106 cells. ABR-001 gene-editing technology was developed by Arbor
Biotechnologies, Inc54. Following electroporation, T cells were transduced with AAV at
an MOI of 15,000 and incubated at 37°C. For further expansion, cells were
supplemented with IL-2 every 2-3 days. At day 14 post-activation, TCRα/β depletion
was performed using EasySep™ human TCRα/β depletion kit (STEMCELL
Technologies,17847) as instructed by the manufacturer’s protocol. T cells were
cryopreserved in 90% FBS/10% DMSO or CS5 freezing media (STEMCELL
Technologies, 07953) using rate-controlled freezing chambers (Corning, CLS432007)
and stored in liquid nitrogen vapor phase.

Cytotoxicity and cytokine-release assay
For short-term cytotoxicity assays, CAR T cells were co-cultured with luc-GFP-
expressing Raji cells or with primary target cellsP at defined E:T ratios for 24 hours in
RPMI 1640 medium (Gibco, 72400-47) supplemented with 10% FBS (Cytiva,
SH300.70.03). Target cell killing was determined using Bright-glo reagent (Promega,
E2650) or flow cytometric analyCsis. For cytokine-release assays, CAR T cells were co-
cultured with Raji target ceTlls at an E:T ratio of 1:4. At 24 hours, cell culture supernatant
was assayed for cytokines using the MSD U-PLEX platform (Meso Scale Diagnostics,
K15049K-2). For long-term cytotoxicity assays, CAR T cells were co-cultured with
luciferase-GFP-expressing Raji cells at an initial E:T ratio of 8:1 for 48 hours in RPMI
1640 medium supplemented with 10% FBS. Half of each well was then removed to
measure target cell killing as determined using Bright-glo reagent. The remaining cells
of each well were passaged on to fresh Raji cells and this process was repeated every
2-3 days until cytotoxic activity was no longer detected. The percentage killing was
determined relative to target-only control wells.

Flow cytometry, staining, and antibodies
Mouse peripheral blood, bone marrow, and spleen samples were lysed using ACK lysing
buffer (Gibco, A1049201). Samples for flow cytometric analyses were stained on ice with
Fixable Viability Ghost Dye (Tanbo, 13-0865-T100) before cell surface staining. An anti-
idiotype antibody against the CD70 scFv was raised in Balb/c mice and was purified from
hybridoma cell culture supernatants. Antibodies were labeled with R-PE (Prozyme,
PJRC10-05) using sulfhydryl-maleimide reaction chemistry. For p-p38 MAPK staining,
cells were stimulated with either PMA and ionomycinS (eBioscience, 00-4970-93) or
CD70KO Raji target cells at an E:T ratio of 1:2 for 20R minutes and immediately fixed for 10
minutes at 37°C prior to intracellular staining. C ells were fixed in BD Cytofix fixation buffer
(BD Biosciences, 554714) prior to analysiIs in a CytoFLEX LX flow cytometer. CAR T cells
were enumerated using 123count eBeads counting beads™ (Invitrogen, 01-1234-42).
Flow cytometric analyses were performed using FlowJo software.

Mixed lymphocyte reaction (MLR) assays
For PBMC MLR assays, host PBMCs were co-cultured with TRACKO graft T cells at a
ratio of 10:1 in 200 µL RPMI medium supplemented with 10% FBS and 20 IU/mL of
recombinant human IL-2 in round-bottomed 96-well plates. For alloreactive T cell MLR
assays, unedited graft donor T cells were irradiated at 30 Gy and co-cultured with host
PBMCs at a ratio of 1:1 in RPMI supplemented with 10% FBS and 20 IU/mL of
recombinant human IL-2, IL-7 (Miltenyi Biotec, 130-093-937), and IL-15 (Miltenyi Biotec,
130-093-955). On day 4 of the co-culture, 50% of the cell culture medium was replaced
with fresh RPMI supplemented with 10% FBS. On day 7, primed T cells were isolated
using human T cell isolation kit (Miltenyi Biotec, 130-096-535) as instructed by the
manufacturer’s protocol. Primed alloreactive T cells were then co-cultured with TRACKO
graft T cells at a 1:1 ratio in 200 µL RPMI medium supplemented with 10% FBS and 20
IU/mL of recombinant human IL-2 in round-bottomed 96-well plates. If MLR co-cultures
exceeded 4 days, 50% of the medium was replaced on day 4. Medium was then
replaced every 2-3 days after. Cells were analyzed by flow cytometry at the indicated
time points. Graft CAR T and host cells were differentiated based on expression of HLA-
A2 (Supplementary Fig. 13).

Antigen escape assays
To model antigenic tumor heterogenEeity, CD19KO and CD70KO target cells were first
mixed at 1:1 ratio and then co-cCultured with CAR T cells at an E:T ratio of 1:1 or 1:4.
Cells were cultured in RPMTI medium supplemented with 10% FBS in 96-well or 24-well
G-Rex plates (Wilson Wolf, 80192M). On days 2, 50% of the cell culture medium was
replaced with fresh RPMI supplemented with 10% FBS. At the indicated time points,
100 µL samples of the heterogeneous cell mixtures were removed and analyzed by flow
cytometry. Target cells were enumerated using 123count eBeads counting beads™
(Invitrogen, 01-1234-42).

Virus-specific T cell tri-culture
Virus-specific T cells (VSTs) were purchased from STEMCELL Technologies (70024)
and autologous B-lymphoblastoid cell lines (BLCLs) were purchased from Charles River
Laboratories (5819750-30). CD19/CD70 double knockout (dKO) BLCLs, herein referred
to as antigen presenting cells (APCs), were generated as described above. APCs were
incubated with 1 µg/mL of pp65 peptides (Charles River Laboratories, 5819750-10) for 1
h at 37°C. Peptide-pulsed APCs were washed extensively prior to tri-culture with VSTs
and CAR T cells. Surviving cells in tri-cultures were quantified by flow cytometry at 48 h.

Orthotopic mouse model
All procedures performed on animals were reviewed and approved by the IACUC of
Allogene Therapeutics, with protocol review faciPlitated by Advanced Bioresources, and
were conducted in accordance with institutional and national guidelines for animal care
and use. Animals were maintained inE a high-barrier, specific pathogen-free animal
facility with a 12‑h light/12‑h daCrk cycle, at an ambient temperature of 20–26 °C and
relative humidity of 30–70%T. Food and water were provided ad libitum. Female NSG
mice (Strain: 005557) 8 to 12 weeks of age were obtained from The Jackson
Laboratory. Experimental and control mice were housed separately. Parental, CD70KO,
or a 1:1 mixture of CD19KO and CD70KO luciferase-GFP-expressing Raji cells were
prepared, and 1 x 105 cells were administered to mice by i.v. injection. Tumor burden
was measured using an IVIS Spectrum instrument (PerkinElmer) twice weekly. Mice
were randomized based on total body bioluminescence 3 days after tumor cell injection.
CAR T cells were injected immediately after thawing, 4 days after tumor cell injection.
To monitor CAR T expansion following administration, peripheral blood was collected
into EDTA-coated tubes (BD, 365974) using submandibular or retro-orbital bleeding at
indicated time points and 20 µL was used for flow cytometric analyses. Blood samples
were subjected to red blood cell lysis twice using ACK lysing buffer (Gibco, A1049201)
and then incubated with Fc Block (TruStain FcX™ PLUS, Biolegend, 156604) for 15 min
at 4 °C, followed by surface antibody staining. Absolute numbers of CAR T cells were
quantified using 123count eBeads (Invitrogen, 01-1234-42). Mice were euthanized by
CO inhalation followed by cervical dislocation as a secondary method when disease
2
model-specific endpoints were exhibited, including hind-leg paralysis, tumor
bioluminescence exceeding 1x1010 photons/s, ruffled fur, or 20% reduction in body
weight.

Humanized mouse models
Female Hu-CD34 mice (Strain: 7055E57), aged 16 to 17 weeks, were obtained from The
Jackson Laboratory. Hu-CD34 Cmice received a single dose of CAR T cells or non-
transduced control T cells Tvia i.v. injection. Human B and T cells and CAR+ cells were
monitored in peripheral blood by flow cytometric analysis weekly. For the PBMC mouse
model, female NSG mice (Strain: 005557) aged 7 to 8 weeks were pre-conditioned with
1 Gy irradiation and 1 x 105 (LD condition only) or 1-2 x 107 human PBMCs isolated
from healthy donors or SLE donors were adoptively transferred via i.v. injection. Three
or seven days after PBMC transfer, mice were randomized and received a single dose
of CAR T cells via i.v. injection or were left untreated. Cells from spleen and blood were
analyzed by flow cytometry at indicated time points post treatment. Engrafted B and T
cells and total CAR+ cells were assessed and quantified using 123count eBeads
(Invitrogen, 01-1234-42). Human α-dsDNA antibody, IgG, and IgM levels in the plasma
were measured by enzyme-linked immunosorbent assays (Anti-dsDNA ELISA Kit,
Eagle Biosciences, 4015; Human IgG Total ELISA Kit, Invitrogen, BMS2091; Human
IgM Total ELISA Kit, Invitrogen, BMS2098).

Statistical analysis
Data are shown as mean ± Standard Error of the Mean (SEM). The data were analyzed
with an unpaired two-tailed Student t test or one-way and two-way ANOVA followed by
Dunnett’s or Tukey’s multiple comparisons test using Prism GraphPad software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The concept of utilizing dual-targeted CAR T cells (CD19/CD70) to mitigate rejection and antigen escape is certainly intriguing; however, there are several significant concerns that currently temper my enthusiasm. I have outlined these below for clarity.\n1.CD70 is an attractive therapeutic target in autoimmune diseases including SLE, RA, MS and others, due to its potential involvement in disease pathogenesis. In this study, the addition of a CD70-specific CAR was initially presented as a strategy to mitigate immune rejection of conventional anti-CD19 CAR-T cells. However, the primary role of the CD70 CAR is to directly deplete host immune cells, including T cells and NK cells. Thus, rather than viewing this design solely as a means of reducing immune rejection, it is more accurate to describe the dual CAR-T approach as a multi-target cytotoxic strategy aimed at simultaneously eliminating B cells, T cells, NK cells, and possibly other immune subsets. While this broad cytotoxicity can indeed help reduce immune-mediated rejection, it also raises important safety concerns regarding off-target effects and immune homeostasis.\n2.Did the authors knock out CD70 in the dual-target CAR-T cells? If not, it raises the possibility that these cells might engage in fratricide by targeting each other due to shared CD70 expression.\n3.A dual-target CAR-T strategy targeting CD19 and CD70 has already been reported in a prior clinical study (DOI: 10.1182/blood-2023-186888) , which the authors have not cited. Importantly, that study has already progressed to the clinical stage, whereas the current work remains at the preclinical level. Without clearly articulating how this approach differs from or improves upon the existing clinical strategy, the manuscript lacks a compelling rationale for novelty. The authors should clearly explain what unique advantages their system offers, to distinguish it from the already published clinical approach.\n4.Figure 1 primarily demonstrates the cytotoxic capacity of CD70-CAR T cells, rather than supporting the claim that “CD70-CAR T cells show resistance to rejection in vitro.” The authors should consider rephrasing this statement.\n5.In Figures 1C, 1D, and 1H, there is a substantial reduction in CD70⁺ T and NK cells, which the authors attribute primarily to selective depletion by CD70 CAR-T cells. However, it is important to note that CD70 is an activation-induced marker, and its expression is highly dynamic-dependent on stimulation strength, duration, and cellular context. Could alternative explanations such as downregulation of CD70 expression or activation-induced exhaustion account for the observed decrease? Have the authors assessed CD70 expression longitudinally over time to distinguish between cell elimination and phenotypic modulation?\n6.Figure 2 appears somewhat conceptually disorganized, and the rationale for selecting the CD70z CAR variant as the optimized construct remains insufficiently substantiated. While Figures 2B and 2C show that CD70z and Qz variants exhibit higher CAR expression levels, the authors appear to directly correlate this with enhanced anti-rejection activity. However, the functional data in Figures 2F and 2G indicate that the anti-rejection efficacy of CD70z is largely comparable to the original QR3bbz construct, raising concerns about potential overinterpretation of CAR expression as a proxy for functional performance. Moreover, whether high CAR expression could potentially lead to unwanted activation or T cell exhaustion? which may compromise long-term efficacy or safety. Also, the decision to prioritize CD70z over the original QR3bbz CAR seems to rest mainly on higher expression and more favorable differentiation phenotypes, rather than demonstrably superior function. Without clearer evidence of functional or translational advantages, the rationale for selecting CD70z as the lead candidate remains unconvincing.\n7.Although the authors demonstrate that CD70z and CD19 CARs can maintain independent functions, the supporting evidence is limited to in vitro cytotoxicity assays and parallel group comparisons. This leaves potential concerns unaddressed, such as signaling interference, competition for cellular machinery, or spatial constraints associated with dual CAR expression.\n8.In Figure 3E, the CD19/CD70 dual CAR-T cells show slightly reduced antitumor efficacy in the CD70KO Raji model, yet the authors do not explore possible underlying mechanisms. Could this be due to signal diversion by the CD70z CAR, or perhaps activation-induced exhaustion? While CD19 CAR function appears intact, the mechanistic basis for this observation remains unclear. In Figure 3F, CD19/CD70 CAR-T cells demonstrate significantly greater peripheral expansion, which the authors interpret as enhanced efficacy. However, such an interpretation should be approached with caution, as increased expansion does not necessarily equate to improved function. It may instead reflect immune space clearance, compensatory proliferation, or heightened activation.\n9.Finally, potential safety concerns associated with CD70 expressions in normal tissues are not discussed. This is particularly important in the dual-CAR context, where the risk of off-tumor toxicity may be amplified, and functional control of the dual-targeted CAR-T cells could become more challenging.\n10.The authors propose that CD70 can be used to simultaneously eliminate autoreactive B and T cells; however, the manuscript does not provide sufficient evidence demonstrating the stable presence of CD70⁺ T cells in SLE patients. While the figures show that CD70 CAR-T cells can effectively deplete CD70⁺ cells, there is a lack of supporting data confirming sustained CD70 expression under human autoimmune pathological conditions. For instance, evaluating the dynamics of CD70 expressions on T cells from SLE patient-derived PBMCs at various time points and activation states would help validate the assumption that CD70 is a consistent and reliable therapeutic target.\n11.Additionally, CD19/CD70 CAR-T cells nearly disappear by day 30, indicating limited persistence that is not superior to existing allogeneic CAR-T platforms. This diminishes the perceived advantage of targeting CD70 in terms of longevity or durability of response.\n12.The reduction in IgG/IgM levels is presented as a marker of therapeutic efficacy; however, rapid clearance of immunoglobulins by dual CAR-T cells should not be used as a sole efficacy endpoint, especially without addressing the potential risks associated with systemic immunosuppression.\n13.While GvHD was observed in mice treated with CD19 CAR-T cells and not in those receiving CD19/CD70 CAR-T cells, the authors attribute this to the CD70 CAR eliminating reactive host T cells. However, no mechanistic data are provided to track GvHD-related cell populations or to validate this interpretation. Further investigation is needed to substantiate the claim that CD70 CAR prevents GvHD through selective depletion of alloreactive T cells.", "reviewer_2": "In this manuscript, Zhang et al. have established a CD70/CD19 CAR to target both CD19+ B cells and CD70+ alloreactive lymphocytes, to reduce alloreactivity as seen with conventional CD19 CAR T cells. This would circumvent the need for lymphodepletion strategies, while increasing CAR T cell persistence. The authors show an effective depletion of alloreactive T and NK cells following treatment with their bispecific CAR T cells in the context of B cell lymphomas as well as auto-immune diseases, highlighting the potential clinical value of this study. This study is coherently written and addresses an important hurdle for CAR T cell treatment.\nThere are several points that need to be addressed:\n- CD70 is also expressed by dendritic cells upon activation, but potential depletion is not studied. An experiment in which depletion of different DC subsets across organs is examined should be included.\n- As the CD19/CD70 CARs would potentially circumvent the need for rigorous lymphodepletion strategies, it would greatly benefit the manuscript if engraftment of CAR T cells is studied with/without prior lymphodepletion. This would also provide insight in the level of protection from alloreactivity CD70/CD19 bispecificity offers over CD19 conventional CAR T cells.\n- In line with this, as CD70/CD19 dual-specific CARs would overcome the need for lymphodepletion pre-treatment strategies, it is unclear why the authors do perform lymphodepletion in the experiments shown in figure 6&7. Please discuss or repeat without lymphodepletion.\n- In Fig. 3E&F CD70/CD19 CARs had a decreased protective function against CD70KO Raji cells compared to CD19 CARs. However, in this setting they did show enhanced peripheral expansion and in Fig. 3A they show the in vitro killing capacity is not reduced. Please discuss this discrepancy.\n- Figures S4C and S4D do not match figure legends nor what is described in the manuscript text. S4D is missing entirely, please correct."}, "author_response": "We appreciate your thoughtful feedback and the opportunity to clarify the rationale behind our dual-targeted CAR T cell strategy and the broader implications of targeting CD70.\nImportantly, CD70 expression is typically transient and restricted to highly activated immune cells, including subsets of T cells, NK cells, and dendritic cells (Nolt et al., Immunol Rev., 2009). Therefore, the majority of host immune cells are expected to remain unaffected by CD19/CD70 CAR T cells. Moreover, lymphodepletion regimens administered prior to CAR T cell infusion will contribute substantially to reduce T, B, and NK cells, independent of CAR specificity.\nData from our ongoing clinical trial in renal cell carcinoma (TRAVERSE Study, NCT04696731) support the specificity and safety of CD70 CAR T cells. Despite robust expansion of CD70 CAR T cells, only a fraction of peripheral T cells were found to upregulate CD70 and were consequently depleted. Importantly, CD70+ T cells recover as CD70 CAR T cells contract (Srour et al., J Clin Oncol, 2025; example below in comment 11).\nRegarding off-tumor effects, we have not observed non-specific cytotoxicity attributable to CD70 CAR T cells in preclinical models (Panowski et al., Cancer Res., 2020). The CD19 CAR utilizes the clinically validated FMC63 scFv, and the CD70 CAR demonstrates specific binding to CD70, minimizing off-target risks. The safety profile of this dual CAR T cell approach is therefore supported by both preclinical and early clinical data.\nAs with any investigational therapy, the safety and efficacy of CD19/CD70 dual CAR T cells will be rigorously evaluated in clinical trials, with appropriate mitigation strategies in place. Given the contribution of both lymphodepleting regimens and CD70 targeting to host immune cell depletion and recovery, such strategies may include lowering the intensity or the composition of the lymphodepletion regimen, as being currently pursued in clinical studies for other CAR T cell products.\n\nThank you for raising this important point regarding potential fratricide. This concern was carefully considered during the early CD70 CAR development and selection of the anti-CD70 scFv used in our study. We did not knockout CD70 in the dual CAR T cells. The expression of this particular CD70 CAR on T cells leads to cis masking, where the CAR binds to CD70 on the same cell, effectively protecting the cells from fratricide. This mechanism has been previously characterized (Panowski et al., Cancer Research, 2022), and while we cannot entirely exclude the possibility of some fratricide, the observed reduction due to cis masking enables the generation of a functional CD70 CAR T cell product with demonstrated activity in both preclinical models and patients with RCC (Srour et al., 2025). Notably, the frequencies of CD70 CAR T cells reached in peripheral blood in RCC patients treated in the TRAVERSE trial frequently exceeded peak levels achieved by autologous products for hematologic cancers (Srour et al, 2025; Neelapu et al, 2017), further supporting the lack of significant fratricide with this construct.\nWe have added some of these additional points, references, and data to the revised manuscript. This includes production yields of dual CAR T cells, which show no significant compromise, suggesting minimal impact of fratricide. These data are now included in Fig. S5D-E.\n\nThank you for pointing out the previous study reporting the dual-target CAR T cell strategy. The referenced clinical study (DOI: 10.1182/blood-2023-186888) describes an autologous approach in which separately manufactured CD19 and CD70 CAR T cells were co-administered to treat relapsed and refractory primary central nervous system diffuse large B-cell lymphoma. While the initial case report (Tu et al., Front Oncol., 2019) was cited in our manuscript, we have now added this distinction more explicitly.\nIn contrast, our work describes a single, allogeneic “off-the-shelf” CAR T cell product that co-expresses both CD19 and CD70 CARs within the same cell. This design offers several key advantages over the previously published autologous strategy:\n• Manufacturing Efficiency: By integrating both CARs into a single allogeneic product, our approach streamlines production and reduces variability associated with separate cell preparations.\n• Scalability and Accessibility: The off-the-shelf nature of our product enables broader clinical deployment and faster treatment timelines.\n• Expanded Indications: this single CD19/CD70 Dual CAR T cell product is designed for use in a range of hematologic malignancies characterized by CD19 and CD70 expression and in autoimmune diseases. A dual CAR T cell therapy based on this design recently received FDA clearance for clinical evaluation in systemic lupus erythematosus, including lupus nephritis, idiopathic inflammatory myopathies, and systemic sclerosis (NCT07085104).\nWe have updated the manuscript to better highlight the novelty and rationale of this dual CAR T cell strategy for oncology and AID.\n\nWe have revised the statement to more accurately reflect the data shown in Fig. 1, now describing that CD70 CAR T cells eliminate CD70+ lymphocytes and persist in an in vitro rejection model. We have also updated the manuscript to rephrase our use of “resistance” throughout, in line with your feedback.\n\nWe agree that CD70 is an activation-induced marker with a dynamic expression pattern that is also context-dependent. We performed longitudinal assessments of CD70 expression on host PBMCs stimulated with allogeneic TRAC KO T cells. CD70 was initially absent but became upregulated on host T cells, peaking around day 7 and remaining detectable through day 14. These data are presented in Fig.S1. We also added analyses of CD70 expression on NK cells from this experiment (Fig. S1D).\nWhile downregulation of CD70 may contribute to the observed decrease in CD70 cells, our data also show a reduction in total host T cell counts when co-cultured with CD70 CAR T cells, suggesting active elimination. We have included these findings in Fig. S3to help distinguish between cytotoxic depletion and modulation of CD70 expression.\n\nThank you for the insightful feedback. We recognize that our rationale for selecting the CD70z CAR could have been more clearly articulated. From a clinical manufacturing perspective, achieving a higher frequency of CAR+ cells is important for dose consistency and scalability. Most importantly, a key consideration in prioritizing CD70z was the removal of rituximab (R) recognition domains that are present in the QR3bbz construct, which will enable the use of CD19/CD70 Dual CAR T cells in the treatment of patients who have received or may receive rituximab, including patients with non-Hodgkin lymphoma and autoimmune diseases. Equivalent anti-rejection activity, improved CAR expression and CAR T cell phenotype, and eliminating concerns associated with rituximab use led to the selection of the CD70z CAR. We have added this clarification to the manuscript.\n\nThank you for raising this important point. To assess potential spatial constraints that may result from co-expressing two different CARs, we evaluated CD19 antigen binding by flow cytometry. We found that binding of soluble CD19 is similar between single CD19 CAR T cells and dual CD19/CD70 CAR T cells (Fig.S5F-G). We also examined potential signaling interference by measuring p38 MAPK phosphorylation following CD19-specific stimulation. The proportion of p-p38 CAR cells was comparable between single and dual CAR T cells (Fig. S5H-I), in fact slightly higher in the dual CAR T cells, an observation that may relate to the point raised in Comment 8.\n\nThank you for raising these thoughtful points. We have updated our interpretation of the expansion data in Fig. 3F (now Fig. 3D) to reflect that increased proliferation may not directly indicate enhanced efficacy and could be influenced by factors such as activation.\nRegarding the reduced antitumor activity in the CD70KO Raji model, we explored the possibility that heterodimerization of the CD19 CAR and the CD70z CAR could lead to excessive activation. To test this, we engineered T cells with a CD70z CAR containing a CD28 hinge, designed to prevent heterodimer formation with the CD8α-hinged CD19 CAR (Hirabayashi, Nature Cancer, 2021). Comparative analyses showed that uncoupling the CARs did not impair CAR expression, cytotoxicity, or host CD70+ T cell depletion. However, in vivo expansion was significantly reduced, suggesting that heterodimerization of the CD19 and CD70z CARs via the CD8α-hinge may enhance proliferation without significantly affecting cytotoxic function. These findings are now included in Fig. S8.\n\nThank you for raising this important safety concern. We previously evaluated binding of the anti-CD70 scFv in a panel of 36 human tissues (Panowski et al., Cancer Research, 2022), observing limited membrane staining on lymphoid cells (expected) and cytoplasmic staining in thymus and kidney epithelial cells, likely reflecting intracellular CD70 which is not accessible to CAR T cells. Flow cytometry analyses confirmed no surface CD70 expression in healthy kidney samples or cytotoxic activity against this tissue.\nSafety studies in cynomolgus monkeys using a CD70-CD3 bispecific antibody with the same anti-CD70 binder did not reveal any unexpected findings and showed CRS and depletion of mononuclear cells in lymphoid tissues, consistent with the mechanism of action of a CD3-CD70 bispecific antibody.\nAdditionally, clinical data from our group and others (Srour et al., 2025; Pal et al., Cancer Discovery, 2024) have not reported on-target off-tumor toxicity beyond expected transient lymphocyte depletion, as pointed out above. These preclinical and clinical data have supported the approval of an Investigational New Drug (IND) application for a rheumatology basket trial using the CD19/CD70 dual CAR T cell product, and a clinical study is now underway (NCT07085104). These updates and a reference to the limited CD70 expression in normal human tissues have been added to the revised manuscript.\n\nThank you for highlighting this important point. To assess CD70 expression dynamics in T cells from lupus patients, we stimulated patient-derived PBMCs with PMA/ionomycin and observed CD70 upregulation, with levels increasing over time (Fig. S2A). We also tested stimulation with Poly (I:C) but did not observe CD70 induction. Additionally, CD70 was upregulated following priming with allogeneic T cells (Fig. S6D), supporting its relevance as a target under inflammatory conditions.\n\nThank you for pointing this out. The limited persistence observed by day 30 reflects the limitations of the humanized mouse models, which we used primarily to demonstrate in vivo B cell depletion rather than long-term CAR T cell durability. It is also possible that in this model, CAR T cells are present in mouse tissues including spleen or lymph nodes even if they are not detectable by flow cytometry in peripheral blood. The expansion of CAR T cells in peripheral blood of patients with lupus treated with autologous products is typically followed by a contraction phase and reemergence of B cells (Mackensen, Nature Medicine 2022), similar to what we see in this humanized mouse model (Fig. 5).\nAdditional support for the persistence of allogeneic CD70 CAR T cells comes from our ongoing TRAVERSE clinical study in RCC patients, where we observed robust expansion and persistence at levels not typically seen with other allogeneic CAR T cell products (Srour et al., 2025). Allogeneic CD70 CAR T cells were also shown to deplete alloreactive CD70+ host T cells (example below), even at low cell doses (Srour et al., 2025). These findings suggest that CD70-mediated depletion of alloreactive host T cells may contribute to enhance CAR T cell persistence. We have included a highlight of these early clinical results in the revised manuscript.\n\nThank you for this important point. We agree that immunoglobulin reduction alone is not a sufficient marker of efficacy. In our studies, this was used simply as another measure of CAR T cell activity, in addition to the deep depletion of B cells observed in mouse tissues (i.e., spleen), a hallmark of the immune reset seen in clinical trials with autologous CAR T cells (Mackensen, Nature Medicine 2022; Junt, Nature Reviews Immunology, 2025). One caveat with the PBMC-engrafted humanized mouse models is that they do not allow for reconstitution of the naive B cell repertoire that is typically seen in patients following treatment with CD19 CAR T cells. These models are also inadequate to provide information to address potential safety risks associated with immunosuppression, which are typically estimated using data derived from clinical trials. In addition to monitoring the B cell compartment and measuring auto-antibodies in clinical samples, we have incorporated a broader panel of biomarkers to more comprehensively assess safety and efficacy of CD19/CD70 Dual CAR T cells in our ongoing RESOLUTION trial.\n\nThank you for pointing this out. In the manuscript, we noted that some mice treated with CD19 CAR T cells developed GvHD. While it is tempting to speculate that T cells expressing the CD70z CAR may help to mitigate GvHD by depleting xenoreactive host lymphocytes, we did not claim this or a definitive mechanism. We agree that further investigation is needed to validate this hypothesis, and we consider it an interesting direction for future studies.\n\nWe appreciate the reviewer’s perspective on the manuscript and on the potential clinical value of an off-the-shelf CAR T cell product targeting CD19 and CD70. The results described in the manuscript have contributed to support safety and efficacy of CD19/CD70 CAR T cells for the treatment of adults with autoimmune diseases, leading to FDA clearance of a Phase 1 study that is currently recruiting (NCT07085104).\n\nThank you for highlighting this important consideration. We agree that CD70 expression on activated dendritic cells warrants further investigation. As an initial step, we assessed CD70 expression on myeloid cells derived from SLE patient PBMCs following stimulation with Poly (I:C). We did not observe CD70 upregulation under these conditions, and recovery of myeloid cells was limited after 24 hours in the absence of exogenous cytokines. These findings are now included in Fig. S2B.\nWhile it is plausible that CD70 DCs could be targeted by CD70 CAR T cells, we believe that evaluating the functional consequences of such depletion, rather than cell loss alone, would provide more meaningful insights. Unfortunately, modeling this in humanized mice is challenging due to limited immune cell functionality and impaired immune cell crosstalk (Brehm et al., J Infect Dis., 2013). A more suitable approach would involve syngeneic mouse models with intact immune networks, which would require the development and validation of anti-mouse CD70 CARs. However, differences in CD70 expression between mice and humans may limit the translational relevance of such models.\nImportantly, a prior toxicology study in non-human primates using a CD3-CD70 bispecific antibody containing the same binder as our CD70 CAR (Panowski et al., Cancer Res., 2022) reported no microscopic abnormalities in the majority of tissues examined. The observed decrease in lymphoid tissue cellularity was attributed to on-target depletion of CD70 lymphocytes, with no evidence of broader tissue toxicity.\n\nThank you for this suggestion. We repeated the experiment in Fig.7 and included a group where we transferred lower numbers of host PMBCs to represent a lymphodepleted condition. Details and results are discussed in our response to the comment below.\n\nThank you for this important observation. The experiments shown in Fig. 6 and Fig. 7 were conducted in immune-deficient NSG mice, where irradiation was used not for lymphodepletion in the conventional sense, but rather to facilitate engraftment of human PBMCs. This is a standard step in this model and does not reflect a preconditioning regimen analogous to clinical lymphodepletion. Therefore, these studies were designed to model a setting in which CAR T cells are introduced into hosts with functional, HLA-mismatched human immune cells, without prior lymphodepletion.\nTo more directly assess the impact of lymphodepletion, we performed a complementary experiment in which CAR T cells were infused following engraftment of either high (no lymphodepletion) or low (lymphodepletion-like) numbers of host PBMCs. This allowed us to compare CAR T cell persistence under conditions that mimic the presence or absence of lymphodepletion. We observed that CD19 CAR T cells failed to persist in the absence of lymphodepletion, whereas CD19/CD70 CAR T cells did persist. In contrast, both CD19 and CD19/CD70 CAR T cells persisted throughout the duration of the experiment in the lymphodepletion group. These findings further support the ability of CD19/CD70 CAR T cells to overcome immune rejection without the need for pre-treatment lymphodepletion.\nThese new data have been added to Fig. 7F and Fig. 7G, and the manuscript has been updated to clarify the experimental rationale and implications.\n\nThank you for pointing out this discrepancy. The in vivo Raji model is highly aggressive and may amplify subtle differences between CD19 and CD19/CD70 CAR T cells. While we observed slightly reduced protective function in this setting, the differences did not reach statistical significance.\nTo explore the potential mechanism behind the enhanced expansion and modestly reduced efficacy, we investigated whether heterodimerization between the CD19 and CD70z CARs could lead to excessive CD3ζ-mediated activation. These findings are discussed in detail in our response to Comment 8 above and supported by new data shown in Fig. S8.\n\nThank you for catching this. We corrected the issue and have now included the correct figures."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I think the authors have addressed all of my concerns, I dont have further comments.", "reviewer_3": "The authors have, generally, responded to reviewer comments with a few points outlined below.\n1) DC depletion – This is a potential risk but I agree with the reviewers that this is difficult to model and will ultimately will require human data. From a safety perspective, CD70 targeting has been in the clinic.\n2) Circumventing the need for lymphodepletion – this is an interesting potential advantage of the CD70 targeting CAR, perhaps even a more important as CAR T cells expand in AI diseases. The experiment included in revised manuscript (high vs low dose PBMCs) is a reasonable attempt. However, the authors should discuss the limitations of this experiment - that is many of the T cell homeastatic cytokines (IL-7 and IL-15 for example) are not available to lymphocytes in this model (no mouse-human cross reactivity and primarily stromal-cell derived)"}, "author_response": "We agree with the reviewer. Accordingly, we have cited multiple clinical studies in the manuscript (ref. number 19-21, 49) that assess the safety of CD70-directed therapies, providing relevant human safety context for this approach.\n\nWe appreciate the reviewer’s insightful comments and agree that our high-vs-low PBMC experiment has important limitations. These conditions do not fully recapitulate the cytokine milieu present in lymphoreplete patients, where homeostatic cytokines may support CAR T cell expansion. Our data should be interpreted as supporting the concept that CD70 CAR-mediated targeting of activated CD70+ host immune cells can partially mitigate competitive or rejection pressures, which may act synergistically with endogenous homeostatic cytokines in patients.\nNonetheless, the observation that our CD19/CD70 CAR T cells maintained potent activity, showed expansion, and persisted even in a high host PBMC setting, despite suboptimal cytokine milieu, is encouraging. We have clarified this limitation and the interpretation of the findings in the revised Discussion."}]

Decision letter