RAPA-501-Allo Therapy of COVID-19-ARDS

The first-in-human Phase 1 study component will evaluate two dose levels of RAPA-501-ALLO off
the shelf cells in patients with COVID-19-related ARDS, with key endpoints of safety,
biologic and potential disease-modifying effects. The randomized, double-blind,
placebo-controlled Phase 2b study component will evaluate infusion of RAPA-501 ALLO off the
shelf cells or a control infusion, with the primary endpoint assessing whether RAPA-501 cells
reduce 30-day mortality.

The COVID-19 pandemic is a disaster playing out with progressive morbidity and mortality. As
of April 6th, 2021, an estimated 132.1 million people have contracted the virus and 2,866,000
deaths have resulted globally. The United States has the highest totals with an estimated
30.8 million people diagnosed and 556,000 deaths. The United States has the highest totals
with an estimated 22.4 million people diagnosed and 375,000 deaths. In stages 1 and 2 of
COVID-19, viral propagation within the patient is predominant. As such, therapeutic
interventions focus on immune molecules (convalescent serum, monoclonal antibodies) and
anti-viral medications (remdesivir). In marked contrast, the most severe and deadly form of
COVID-19, stage 3, is driven not by viral propagation, but by an out-of-control immune
response (hyperinflammation) caused by increases in immune molecules known as cytokines and
chemokines. As such, therapeutic interventions for stage 3 disease focus on anti-inflammatory
medications such as anti-cytokine therapy (anti-IL-6 drugs) or corticosteroid therapy.
Unfortunately, such interventions do not address the full pathogenesis of stage 3 COVID-19,
which includes hyperinflammation due to "cytokine storm" and "chemokine storm," tissue
damage, hypercoagulation, and multi-organ failure (including lung, heart, kidney and brain).
The pulmonary component of stage 3 disease includes acute respiratory distress syndrome
(ARDS), which is a final-common-pathway of patient death due to a myriad of conditions,
including pneumonia, sepsis, and trauma. There is a dire need for novel cellular treatments
that can deliver both a broad-based immune modulation effect and a tissue regenerative
effect, such as RAPA-501-ALLO off-the-shelf allogeneic hybrid TREG/Th2 Cells.

The mechanism of fatal pneumonia in experimental murine coronavirus infection is mediated by
innate inflammasome activation, a resultant robust infiltration of inflammatory monocytes and
macrophages, and a subsequent increase in multiple pro-inflammatory cytokines and chemokines.
Importantly, the CD4+ and CD8+ adaptive T cell response to experimental coronavirus infection
can be either curative or disease propagating. Viral-induced pulmonary inflammation has also
been evaluated in non-human primate models, which have confirmed that dysregulated immune
activation during viral pneumonia involves T cell subset imbalance, a downstream
monocyte-derived inflammatory cascade, and resultant epithelial cell injury. In humans,
severity of viral-induced lower respiratory tract infection correlates with increased numbers
of effector memory CD8+ T cells in the airway. As recently reviewed, human coronavirus
infection represents an ongoing battle between virus and host, with the nature of the
response dictating disease cure or alternatively, aggravated lung disease. Evidence exists
that the adaptive immune system contributes to pulmonary inflammation during SARS-associated
Coronavirus (SARS-CoV) disease: that is, bronchoalveolar fluid from such subjects had
increased T cell numbers and increased Th1-associated molecules IL-12, IFN-gamma, and IP-10.
This downstream adaptive T cell-mediated inflammatory response is driven in part by the
SARS-Co-V protein viroporin 3a. Specifically, viroporin molecules activate the NLRP3
inflammasome that links innate-to-adaptive inflammatory responses. Furthermore, both SARS-CoV
and SARS-CoV-2 express an open-reading-frame ORF3a molecule, which also activate the NLRP3
inflammasome.

The nature of immunity during resolution of symptomatic COVID-19 infection has recently been
reported, namely: emergence of antibody-secreting cells and CD4+ T follicular helper cells;
increase in perforin- and granzyme-expressing CD8+ T cells; and relative lack of an increase
in pro-inflammatory cytokines and chemokines. In marked contrast, patients with severe
COVID-19 disease had greatly increased plasma levels of cytokines (including IL-2 and
TNF-alpha) and chemokines (including IP-10 and MIP-1-alpha) (Huang et al., Lancet, 2020).
Collectively, these results indicate that pro-inflammatory cytokine and chemokine responses
that occur in severe COVID-19 is detrimental and that effective anti-inflammatory approaches
may ultimately prove to be therapeutic. However, as previously detailed, the more advanced
Stage 3 COVID-19 disease is characterized by an ARDS component, and cytokine storm, as such,
novel approaches to treat the COVID-19 viral pneumonia should optimally incorporate both an
anti-inflammatory element and a tissue protection/tissue repair element.

Stage 3 COVID-19 carries an estimated 30-day mortality of over 50% in spite of ICU
utilization, mechanical ventilation, and supportive care therapies to manage ARDS and
multiorgan failure. Narrowly acting targeted anti-inflammatory approaches such as anti-IL-6
therapeutics have not been particularly effective in stage 3 COVID-19 and the broad
anti-inflammatory pharmaceutical approach of corticosteroid therapy, has only modestly
tempered stage 3 disease in some studies. Cell therapy is also being evaluated in stage 3
COVID-19, in particular, mesenchymal stromal cells (MSC) and now, with the current
RAPA-501-ALLO protocol, regulatory T (TREG) cells. TREG therapy has a mechanism of action
that includes a multi-faceted anti-inflammatory effect, which puts TREG therapy at the
forefront of future curative therapy of a wide range of autoimmune and neurodegenerative
diseases, plus transplant complications, such as graft-versus-host disease (GVHD) and graft
rejection. In addition, TREG therapy can provide a tissue regenerative effect, which places
TREG cell therapy at the lead of novel regenerative medicine efforts to repair a myriad of
tissue-based diseases, such as diseases of the skin, muscle, lung, liver, intestine, heart
(myocardial infarction) and brain (stroke). RAPA-501-ALLO off-the-shelf cell therapy offers
this potential dual threat mechanism of action that incorporates both anti-inflammatory and
tissue repair effects for effective treatment of COVID-19 and multiple lethal conditions.

RAPA-501-ALLO cells are generated from healthy volunteers, cryopreserved, banked, and are
then available for off-the-shelf therapy anytime. During manufacturing, T cells are
"reprogrammed" ex vivo using a novel, patented 7-day two-step process that involves T cell
de-differentiation and subsequent re-differentiation towards the two key anti-inflammatory
programs, the TREG and Th2 pathways, thus creating a "hybrid" product. The hybrid phenotype
inhibits inflammatory pathways operational in COVID-19, including modulation of multiple
cytokines and chemokines, which attract inflammatory cells into tissue for initiation of
multi-organ damage. The hybrid TREG and Th2 phenotype of RAPA-501-ALLO cells cross-regulates
Th1 and Th17 populations that initiate hyperinflammation of COVID-19. RAPA-501 immune
modulation occurs in a T cell receptor independent manner, thus permitting off-the-shelf cell
therapy. Finally, in experimental models of viral pneumonia and ARDS, TREG cells mediate a
protective effect on the lung alveolar tissue. Because of this unique mechanism of action
that involves both anti-inflammatory and tissue protective effects, the allogeneic RAPA-501 T
cell product is particularly suited for evaluation in the setting of Stage 3 COVID-19-related
ARDS.

In general, classical autoimmune disease, neurodegenerative disease, and viral-induced
inflammatory disease are driven by predominance of Th1/Th17-type responses with a relative
insufficiency of the counter-regulatory immune suppressive Th2 and TREG subsets. TREG cells,
which are defined in part by their expression of FOXP3 transcription factor, have been
extensively studied in experimental models for their capacity to modulate autoimmune disease,
neurodegenerative disease, and transplantation complications, including graft-versus-host
disease (GVHD) and graft rejection. Importantly, T cell production of IL-2 or exogenous IL-2
administration drives lung inflammation during experimental viral infection; therefore, given
the known role of TREG cells as a consumer of IL-2, there is a strong mechanistic rationale
for a beneficial contribution of TREG cells during viral-driven lung inflammation.
Furthermore, in an experimental murine models of virus-induced lung inflammation and lung
injury, interventions that augmented TREG cell number and function accelerated the repair of
lung injury. And, Th2 cells, which are defined in part by their expression of GATA3
transcription factor, were described thirty years ago as a powerful counter-regulatory
population to prevent Th1 cell predominance. It is important to note that clinical data
indicates that maneuvers that increase immune suppressive cell populations, including TREG
cells, can reduce severe inflammatory disease such as graft-versus-host disease without
impairing anti-viral immunity. In addition, in both experimental models and clinical studies,
an appropriate level of TREG cells can result in an overall improvement in pulmonary
inflammation during viral bronchiolitis. Collectively, these findings indicate that T cells
expressing a combined TREG/Th2 phenotype would predictably yield beneficial effects in the
setting of viral-induced pulmonary inflammation and injury associated with severe Stage 3
COVID-19 disease.

Clinical trials have evaluated both TREG and Th2-type cells for various conditions, most
prominently transplantation complications. In general, thymic-derived natural (n)TREG cells
are thought to express a more stable phenotype than post-thymic induced (i)TREG cells; by
comparison, iTREG cells can mediate more potent suppression. Nonetheless, both nTREG and
iTREG cells are susceptible to differentiation plasticity, which creates concern that a
potentially therapeutic TREG population might convert to pathogenic Th1/Th17 phenotypes in
vivo. Ex vivo expanded (n)TREG cells were evaluated in the setting of allogeneic
hematopoietic cell transplantation (HCT) using cord blood donors; more recently, the same
research group has developed ex vivo expanded (i)TREG cell therapy to limit transplant
complications. In addition, ex vivo expanded nTREG cells have been evaluated in the setting
of type I diabetes mellitus; this clinical trial found that TREG therapy was safe and at
least transiently effective in improving disease control. Furthermore, in the setting of
amyotrophic lateral sclerosis (ALS), which is a disease propagated by a severe Th1-driven
peripheral and central inflammatory response, a clinical trial of nTREG cell adoptive
transfer identified that the intervention was safe and showed promise in terms of ALS disease
amelioration. Finally, in a phase II study of rapamycin-resistant Th2 cell therapy in the
setting of low-intensity allogeneic HCT, adoptive Th2 cell transfer was safe and associated
with a shift towards Th2 polarization in vivo, preservation of donor engraftment,
stabilization of mixed chimerism, a low rate of GVHD, and potent anti-tumor effects in
patients with refractory hematologic malignancy. Collectively, these clinical trial results
indicate that the adoptive transfer of TREG and Th2-type populations can be safely
administered, even in the allogeneic HCT setting, and can mediate beneficial modulation of
inflammatory conditions.

Ex vivo manufacturing can be utilized to generate induced (i)TREG cells from the post-thymic
pool of peripheral T cells. In the current protocol, the manufacturing method will focus upon
mTOR inhibition, which is an established intervention that promotes the induction of TREG
cells. In combination with mTOR inhibition, the culture system that is utilized incorporates
cytokines that promote both a Th2 and a TREG phenotype, namely, IL-4, TGF-beta, and IL-2.
Finally, the protocol will include a TREG/Th2 cell product comprised of both CD4+ and CD8+ T
cell subsets, as these counter-regulatory T cell subsets express differential T cell receptor
(TCR) repertoires and a diversity of effector mechanisms that can potentially enhance an
anti-inflammatory effect. For RAPA-501 manufacture, peripheral blood mononuclear cells are
collected from a steady-state apheresis and subjected to the following two-step culture
intervention: in step 1, a severe starvation step results in T cell de-differentiation, which
is realized through both selective media utilization and the addition of FDA-approved
pharmaceutical agents that potently inhibit mTOR signaling; and in step 2, re-differentiation
of T cells occurs using co-stimulation agents and Th2- and TREG-type polarizing cytokines.
After 6-days in culture, the resultant TREG/Th2 population is cryopreserved in single-use
aliquots at protocol-driven therapeutic doses. T cells of type II cytokine phenotype are
characterized in part by their expression of the transcription factor GATA3 whereas
regulatory T cell populations are identified in part by their expression of FOXP3
transcription factor. At culture initiation, a very low frequency of T cells express either
GATA3 or FOXP3. In contrast, the RAPA-501 cell product manufactured in the TREG/Th2 culture
conditions expresses a high frequency of T cells that are either single-positive for GATA3,
single-positive for FOXP3, or double-positive for both GATA3 and FOXP3. Importantly, this
transcription factor profile is expressed in both manufactured CD4+ and CD8+ T cells. It is
important to note that both CD4+ and CD8+ T cell populations with TREG function have been
defined, including a CD8+ TREG population that potently suppresses GVHD; it is potentially
advantageous to have both subsets represented in a therapeutic product because CD4+ and CD8+
TREG cells utilize differential effector mechanisms.

Regulatory T cell populations can suppress pathogenic effector T cell populations by several
defined mechanisms, including through expression of CD39 and CD73 ectonucleotidase molecules,
which act to hydrolyse pro-inflammatory ATP towards the immune suppressive adenosine
substrate . Indeed, TREG cells that express CD39 possess increased suppressive function and
have been associated with resolution of inflammatory bowel disease. Furthermore, suppressive
function of human TREG cells is mediated in part by CD73. T cells manufactured in the
TREG/Th2 culture condition have an increase in expression of the TREG-associated effector
molecules, CD39 and CD73. In addition to the CD39/CD73 ectonucleotidases, TREG cell function
has also been correlated with expression of CD103, which is an integrin that dictates
epithelial lymphocyte localization. Indeed, CD103 and IL-2 receptor signaling cooperate to
maintain immune tolerance in the gut mucosa; furthermore, CD103-expressing TREG cells are
critical for amelioration of experimental chronic GVHD . T cells manufactured in the TREG/Th2
culture condition have increased expression of the TREG effector molecule CD103.

In experimental models, the efficacy of adoptive T cell therapy is dependent upon successful
engraftment and in vivo T cell persistence. Importantly, the T cell differentiation state
helps dictate in vivo persistence, with less differentiated cells having increased
persistence. Murine rapamycin-resistant T cells, which expressed a T central memory (TCM)
phenotype, had increased in vivo engraftment potential relative to control T cells. In
addition, human rapamycin-resistant T cells also had increased engraftment in a
human-into-murine model of xenogeneic graft-versus-host disease. T cells with reduced
differentiation relative to the T effector memory (TEM) population have increased in vivo
persistence and mediate increased in vivo effects, including the TCM subset, the naïve T cell
subset, and more recently, the T stem cell memory (TSCM) subset. This relationship between T
cell differentiation status and in vivo T cell function is relevant to TREG cells, as: (1)
TREG cells of TCM phenotype were more effective at reducing experimental GVHD relative to
TREG cells of TEM phenotype; and (2) TREG cells that expressed the stem cell marker CD150
were highly effective for the prevention of stem cell graft rejection. T cells manufactured
in the TREG/Th2 culture condition are enriched for cells having a reduced differentiation
state consistent with a T stem cell subset, including expression of the CD150 marker.

It is also important to assess the cytokine secretion profile of the manufactured RAPA-501
cells. First, it is critical that the cell product is capable of IL-4 secretion, which is the
driver cytokine for subsequent Th2 differentiation. Second, it is desirable that an
adoptively transferred T cell population is capable of secreting IL-2, as this capacity
indicates a progenitor function that permits T cells to expand more readily in vivo without
the need for exogenous IL-2 . Finally, it is important that the RAPA-501 cell population has
reduced secretion of the Th1- or Th17-type cytokines IFN-gamma, TNF-alpha, IL-17, and GM-CSF.
The manufactured RAPA-501 cell product secretes IL-4 and IL-2 with minimal secretion of Th1-
or Th17-type cytokines.

Regulatory T cells can also be defined in part by their ability to suppress the proliferation
or function of effector T cells. Importantly, manufactured TREG/Th2 cells potently suppress
Th1/Tc1 cell secretion of multiple inflammatory cytokines, including IFN-gamma, GM-CSF, and
TNF-alpha. To assess the mechanism of suppression, experiments were performed using the
transwell assay, whereby effector T cells and RAPA-501 cells are separated by a filter that
prevents cell-to-cell contact but allows cell communication by small soluble mediators such
as cytokines. RAPA-501 cells acted in a TCR-independent manner to suppress the cytokine
secretion capacity of effector T cells. Because no co-stimulation was provided to the
transwell chamber containing RAPA-501 cells, RAPA-501 cells did not require co-stimulation to
modulate inflammatory cytokine levels, including IL-2, IFN-gamma, GM-CSF, and TNF-alpha. The
following cytokines and chemokines were reduced by RAPA-501 cells in this transwell assay,
with relatively equal suppression mediated by CD4+ and CD8+ subsets of RAPA-501 cells: CCL1,
CCL2, CCL7, CCL11, CCL13, CCL17, CCL20, CCL22, CCL26, CXCL1, CXCL10, CXCL11, CXCL12, IL-6,
IFN-gamma, GM-CSF, and IL-10. The ability of TREG cells to consume IL-2 is a commonly
described phenomenon, although previous studies identified the requirement of cell-to-cell
contact for IL-2 consumption. As such, RAPA-501 cells are uniquely capable of modulating the
level of multiple inflammatory cytokines in a contact-independent manner. These results
suggest that RAPA-501 cells represent a suitable candidate for neutralization of multiple
cytokines and chemokines associated with various diseases, including viral-induced lung
inflammation. In light of this capacity of the RAPA-501 cell product to suppress T
cell-mediated inflammation in a TCR-independent manner, RAPA-501 therapy is highly suitable
for allogeneic, off-the-shelf treatment applications.

Further experiments were performed to evaluate whether the RAPA-501 cell product might also
regulate human CNS microglial cells, which are myeloid-derived cells that are analogous to
the peripheral monocyte population. To evaluate whether the RAPA-501 cell product might
modulate pro-inflammatory microglial cells in a contact-independent manner, the ability of
RAPA-501 cells to reduce the inflammatory state of the microglial cell line HMC3 was tested
in a transwell assay. RAPA-501 cells reduced the HMC3 cell secretion of the pro-inflammatory
cytokines IL-6 and IFN-gamma and the pro-inflammatory chemokine IP-10 at the relatively low
ratio of RAPA-501 cells to microglial cells of 1:40. These results demonstrate that the
RAPA-501 product is capable of inhibiting cytokine and chemokine secretion from
myeloid-derived populations in a contact- and TCR-independent manner, thereby providing a
further rationale for allogeneic, off-the-shelf utilization of the RAPA-501 product.

RAPA-501 cell product for stability of T cell phenotype was also evaluated. That is, it has
been determined that a limiting factor of TREG cell therapy may be differentiation
plasticity, for example, whereby TREG cells can be influenced by inflammatory cytokines to
lose their TREG characteristics and adopt a Th1-type inflammatory state, which may then
promote disease pathogenesis. To address this possibility, the RAPA-501 cell capacity to
modulate T cell differentiation transcription factors after an extended culture interval that
involved T cell co-stimulation, the absence of mTOR inhibitors, and the presence of the
polarizing inflammatory cytokines IFN-alpha and IL-6 was studied. These experiments
demonstrated that the RAPA-501 cell product had remarkable differentiation stability
(continued expression of FOXP3 and GATA3 in CD4+ and CD8+ subsets; lack of up-regulation of
TBET).

In summary, RAPA-501 cells express a phenotype consistent with a regulatory T cell
population, including: stable expression of the TREG and Th2 transcription factors FOXP3 and
GATA3; expression of the TREG functional molecules CD39, CD73, and CD103; expression of a
reduced state of T cell differentiation, including expression of the stem cell marker CD150;
secretion of a Th2 pattern of cytokines with minimal secretion of Th1/Th17-type cytokines;
functional suppressive capacity against both effector Th1/Tc1 cells and pro-inflammatory
myeloid cells, including reduced levels of multiple inflammatory cytokines and chemokines;
and a capacity to inhibit inflammatory effectors in a contact- and TCR-independent manner.
Collectively, these characteristics of the RAPA-501 product predicts that this therapy
represents a novel and promising candidate to treat COVID-19-related ARDS.

This is a first-in-human phase 1/phase 2b study evaluating allogeneic RAPA-501 cell therapy
in participants with COVID-19-related ARDS. Two phase 1 cohorts will be evaluated, namely, a
low-dose Cohort 1 (40 x 10^6 cells/infusion) and a high-dose Cohort 2 (160 x 10^6
cells/infusion); this phase 1 component will utilize dose-limiting-toxicity (DLT) as a
primary endpoint. Provided that safety is demonstrated in the phase 1 study component, each
RAPA-501 dose cohort can be evaluated in the randomized phase 2b component. In the phase 2b
component, for each RAPA-501 dose level determined to be safe, patients will be randomized to
receive RAPA-501 cells (n=19) or placebo (n=19). After infusion of either RAPA-501 cells or
placebo, these randomized cohorts will continue on standard-of-care therapy (not
protocol-driven). The primary endpoint of the phase 2b study is 30-day mortality, with the
statistical goal of reducing mortality in RAPA-501 recipients relative to the randomized,
placebo-control cohort.

Study participants must be hospitalized patients with COVID-19-related ARDS, specifically:
SARS-CoV-2 infection, as determined by RT-PCR or equivalent test; pulmonary infiltrate on
radiologic examination; and a diagnosis of ARDS requiring intensive respiratory support
including non-invasive ventilation such as high-flow nasal cannula or mechanical ventilation.
The study consists of: (1) a study enrollment step (screening); and (2) after eligibility is
confirmed, infusion of RAPA-501 cells will be performed. On Cohort 1, infusion of RAPA-501
cells will be at a dose of 40 x 10^6 cells/infusion; three participants will be initially
treated, with one week separating participants in this phase 1 component to evaluate for DLT,
which will be defined as grade 3 or greater toxicity attributable to RAPA-501 cells within
7-days of infusion. At any point that Cohort 1 therapy is deemed to be safe (either 0/3 or
1/6 having a DLT), then Cohort 1 RAPA-501 therapy can be evaluated in the randomized phase 2b
study component to address the primary study objective relating to 30-day mortality. At any
point that Cohort 1 therapy is deemed to be safe, treatment on Cohort 2 can be initiated,
which will evaluate the infusion of RAPA-501 cells at a dose of 160 x 10^6 cells/infusion. In
the same manner as Cohort 1, if Cohort 2 therapy is deemed to be safe, Cohort 2 RAPA-501
therapy can be evaluated in the randomized phase 2b study component to address the primary
study objective relating to 30-day mortality. The study will be comprised of three main
stages: (1) the above-detailed 30-day interval relating to the primary study objectives; (2)
an extended, total 90-day interval to continue relatively intensive clinical monitoring of
potential efficacy and safety endpoints; and (3) an extended, total 6-month interval to
provide long-term clinical follow-up and to continue to monitor for laboratory parameters
relating to immune and viral endpoints.