Exercise as an Immune Adjuvant for Allogeneic Cell Therapies

Background:

Allogeneic cell therapies encompass various approaches, including donor lymphocyte

infusions (DLI) and engineered immune cell products like chimeric antigen receptor (CAR)

T-cells, gamma delta (γδ) T-cells, cytokine-induced killer (CIK) cells, and

cytokine-induced memory-like natural killer (NK) cells. These therapies are commonly

employed after allogeneic hematopoietic cell transplantation (alloHCT) to prevent or

treat leukemic relapse in high-risk patients. However, while these therapies have shown

potential, the success rates for DLI and expanded cell products remain limited. DLI, in

particular, carries the risk of inducing graft-versus-host disease (GvHD), where donor

T-cells attack healthy tissues, leading to significant morbidity. Furthermore, expanded

cell products face challenges related to manufacturing times, efficacy, and cost, which

can limit their accessibility and effectiveness. Therefore, there is a critical need to

enhance the graft-versus-leukemia/lymphoma (GvL) effects of DLI and improve the efficacy

of expanded cell products to achieve better outcomes for a larger number of patients

without increasing the risk of GvHD, thereby broadening their use in clinical practice.

Exercise has been shown to contribute to lower cancer risk, improve outcomes in cancer

survivors, and act as an adjuvant for several cancer therapies. The present exercise

model involves an acute single bout of moderate to vigorous intensity exercise lasting 20

minutes, which evokes a catecholamine-dependent mobilization and redistribution of

effector lymphocytes (e.g., natural killer cells, γδ T-cells, and CD8+ T-cells). This

response may enhance long-term immunosurveillance by improving the recognition and

destruction of premalignant cells and contributing to the suppression of tumor growth.

The overarching research question is: Can lymphocytes be collected from blood during the

exercise-induced mobilization phase to generate superior cell products for cancer

immunotherapy? The overall vision is to develop exercise-mobilized lymphocytes into a

therapy that increases the efficacy of both DLI and expanded cell products (e.g., CAR

T-cells, γδ T-cells, CIK cells, and cytokine-induced memory-like NK cells) for treating

leukemia/lymphoma relapse. This novel approach, termed "DLI-X," has the potential to

improve a pre-existing therapy for the treatment of blood cancers at minimal cost.

The goals of this proposal are to conduct head-to-head comparisons between standard DLI

and DLI-X, both in vitro and in xenogeneic mouse models engrafted with various human

hematological cancers, and to identify the underlying mechanisms driving the enhanced

anti-leukemia/lymphoma response of DLI-X.

The overarching hypothesis is that DLI-X and the expanded cell products manufactured from

these exercise-mobilized lymphocytes will exhibit enhanced GvL effects against multiple

hematological malignancies compared to standard DLI. These effects will be driven by

β2-adrenergic receptor (β2-AR)-mediated transcriptomic and proteomic changes that promote

target cell recognition and cytotoxicity. Additionally, it is proposed that DLI-X will

improve the efficacy of combination therapies such as blinatumomab, a bi-specific T-cell

engager, and monoclonal antibodies designed to increase antibody-dependent cellular

cytotoxicity (ADCC), thereby enhancing tumor growth suppression and the GvL effects of

DLI.

Specific Aims:

1. Evaluate how acute, single bouts of moderate to vigorous intensity exercise lasting

20 minutes influence the number, phenotype, and molecular characteristics of immune

cells in the blood.

2. Determine whether immune cells collected post-exercise yield superior therapeutic

products compared to those collected under resting conditions from the same donor.

3. Investigate the role of adrenergic receptor signaling in mediating these effects

Procedures:

Healthy participants will be recruited into three distinct arms (cohorts) of this study:

(1) Exercise Cohort; (2) Exercise + Beta Blocker Cohort; and/or (3) Isoproterenol

Infusion Cohort. Participants may enroll in one, two, or all three study arms. The

procedures for each cohort are outlined below.

Exercise Cohort Participants in the Exercise Cohort will be scheduled to visit the

laboratory for three separate sessions between 08:00 and 10:00. During each visit, staff

will confirm adherence to pre-testing guidelines (e.g., 8-12 hours of fasting and no

vigorous physical activity). Any participant who does not meet these guidelines will be

rescheduled.

Exercise Cohort Visit 1: Screening and Graded Exercise Test Time Commitment: 60 minutes

1. Informed Consent: A member of the research team will obtain written informed consent

from the participant.

2. Pre-screening Questionnaire: Participants will complete the AHA/ACSM pre-screening

questionnaire to verify that inclusion and exclusion criteria are met.

3. Anthropometric Measurements: Height and weight will be measured.

4. Blood Samples: A fingerstick capillary blood sample will be collected (using a

sterile spring-loaded lancet) for total cholesterol and fasting glucose

quantification, using an automated handheld analyzer (Cardiocheck).

5. Blood Pressure Measurement: Resting blood pressure will be measured using a manual

or automated blood pressure cuff.

6. Risk Stratification: Participants deemed ineligible after screening will be excluded

from further participation.

7. Graded Exercise Test: Participants will perform a maximal exercise protocol on an

indoor stationary bicycle. They will wear a heart rate monitor and a face mask

connected to a metabolic cart for continuous measurement of heart rate and

respiratory gases. The test will begin at 50 Watts for females and 75 Watts for

males, with power increased by 15 Watts each minute until exhaustion. Maximal oxygen

uptake (VO2max), ventilatory threshold, and peak cycling power will be determined.

Exercise Cohort Visits 2 and 3 Time Commitment: 2 hours per visit Participants will

return to the laboratory 3-10 days after Visit 1 and 7-14 days after Visit 2.

1. Pre-Exercise Procedures: An IV catheter will be inserted into a peripheral arm vein

by a trained phlebotomist. A pre-exercise blood sample (approximately 120 mL) will

be collected.

2. Exercise Protocol: Participants will engage in a 20-minute session of

moderate-to-vigorous cycling exercise at power outputs corresponding to 50%, 60%,

70%, and 80% of their predetermined maximal oxygen uptake (VO2max) for 5-minute

increments. Participants will not be exercised to exhaustion during these trials.

Blood pressure measurements and ratings of perceived exertion will be collected

every 5 minutes during the exercise session and immediately after.

3. Blood Sampling During Exercise: Additional venous blood samples (total volume during

exercise: 80 mL) will be collected through the IV catheter at various stages of the

exercise protocol.

4. Post-Exercise Blood Draw: A final blood draw of 15 mL will be collected 1 hour

post-exercise.

Total Blood Volume: Participants will donate a total of 215 mL of blood (120 mL

pre-exercise, 80 mL during exercise, and 15 mL post-exercise) per visit, for a total of

430ml across both visits. Additionally, several droplets of capillary blood

(approximately 10-20 µL) will be collected during Visit 1 for screening purposes.

The procedures for Visits 2 and 3 will be identical. The rationale for two visits is to

obtain sufficient blood to generate multiple therapeutic cell products from the same

donor. The total time commitment for this cohort is approximately 5 hours.

Exercise + Beta Blocker Cohort

Time Commitment: 21 hours Participants in the Exercise + Beta Blocker Cohort will be

scheduled to visit the laboratory for six separate sessions between 08:00 and 10:00,

which will be spread over 6-10 weeks. During each visit, study staff will confirm

adherence to pre-testing guidelines (e.g., 8-12 hours of fasting and no vigorous physical

activity). Any participant who does not meet these guidelines will be rescheduled.

Exercise + Beta Blocker Cohort Visit 1 - Graded Exercise Test Time Commitment: 60

minutes. Participants will complete a graded exercise test on an indoor stationary

bicycle to determine their maximal oxygen uptake (VO₂max) and peak cycling power. This

test will ensure the appropriate intensity levels for subsequent exercise trials.

Exercise + Beta Blocker Cohort Visits 2-6 - Exercise Trials Time Commitment: 20 hours

The remaining five visits will consist of the main exercise trials, where participants

will undergo the following procedures. There will be a 7-10 day period between each

exercise trial visit to allow for recovery and minimize potential carryover effects from

the drugs administered:

1. Drug Administration: The drug trials will be conducted in a block, randomized

double-blind setting to ensure that neither the experimenter nor the participant

knows which trial is occurring. The randomization will be computed by a member of

the research team not involved in performing the exercise trials. The timing of drug

administration is based on peak plasma concentrations of each drug. At 3 hours, 2

hours, and 1 hour prior to each exercise trial, participants will be administered

either a drug or a placebo pill according to the following outline:

Trial 1: Placebo at all time points Trial 2: Nadolol at 3 hours Placebo at 2 hours

and 1 hour Trial 3: Bisoprolol at 3 hours Placebo at 2 hours and 1 hour Trial 4:

Placebo at 3 hours and 1 hour, Carvedilol at 2 hours Trial 5: Bisoprolol at 3 hours,

Placebo at 2 hours, Roflumilast at 1 hour

2. Pre-Drug Procedures: Prior to the ingestion of the drug or placebo, an IV catheter

will be inserted into a peripheral arm vein by a trained phlebotomist. A pre-drug

blood sample will be collected.

3. Post-Drug, Pre-Exercise Blood Sample: After the drug or placebo has been ingested

and 30 minutes before exercise begins, a post-drug, pre-exercise blood sample will

be collected.

4. Exercise Protocol: Participants will engage in a 20-minute session of

moderate-to-vigorous cycling exercise at power outputs corresponding to 50%, 60%,

70%, and 80% of their predetermined VO₂max for 5-minute increments. Participants

will not be exercised to exhaustion during these trials. Blood pressure measurements

and ratings of perceived exertion will be collected every 5 minutes during the

exercise session and immediately after.

5. Blood Sampling During Exercise: Additional venous blood samples will be collected

through the IV catheter at various time points throughout the exercise protocol.

6. Post-Exercise Recovery Blood Samples: Additional blood samples will be collected

during the recovery phase, at various time points ranging from 5 to 60 minutes

post-exercise.

Total Blood Volume: Participants in this cohort will donate a total of approximately 220

mL of blood per visit. The cumulative total blood volume for this cohort across all six

visits is approximately 1,320 mL.

Isoproterenol Cohort Participants in the Isoproterenol Cohort will be scheduled to visit

the laboratory for three separate sessions between 08:00 and 10:00. During each visit,

study staff will confirm adherence to pre-testing guidelines (e.g., 8-12 hours of fasting

and no vigorous physical activity). Any participant who does not meet these guidelines

will be rescheduled.

Isoproterenol Cohort Visit 1: Screening and Isoproterenol Infusion Time Commitment: 2

hours

1. Informed Consent: A member of the research team will obtain written informed consent

from the participant.

2. Pre-screening Questionnaire: Participants will complete the AHA/ACSM pre-screening

questionnaire to verify that inclusion and exclusion criteria are met.

3. Anthropometric Measurements: Height and weight will be measured.

4. Blood Samples: A fingerstick capillary blood sample will be collected (using a

sterile spring-loaded lancet) for total cholesterol and fasting glucose

quantification, using an automated handheld analyzer (Cardiocheck).

5. Blood Pressure Measurement: Resting blood pressure will be measured using a manual

or automated blood pressure cuff.

6. Risk Stratification: Participants deemed ineligible after screening will be excluded

from further participation.

7. Pre-Infusion Procedures: The participant will be fitted with electrodes attached to

an electrocardiogram (ECG; 12-lead) and will receive a resting ECG reading, which

will be monitored by a physician cardiologist. The cardiologist will make the

decision whether to proceed with the infusion or exclude the participant from the

study. Two IV catheters will be inserted into bilateral peripheral arm veins by a

trained phlebotomist. One catheter will be used for delivering isoproterenol, and

one will be used for collecting blood samples. A pre-infusion blood sample

(approximately 120 mL) will be collected.

8. Isoproterenol Infusion Protocol: Participants will receive a continuous infusion of

isoproterenol for 20-minutes at a concentration of 50ng/min/kg. Blood pressure

measurements will be collected every 5 minutes during the infusion and ECG activity

will be monitored continuously. A physician cardiologist will monitor the entire

infusion procedure.

9. Blood Sampling During Infusion: Additional venous blood samples (total volume during

exercise: 80 mL) will be collected through the IV catheter during the last 5-minutes

of the infusion protocol.

10. Post-Infusion Blood Draw: A final blood draw of 15 mL will be collected 1 hour

post-exercise.

Total Blood Volume: Participants will donate a total of 215 mL of blood (120 mL

pre-infusion, 80 mL during infusion, and 15 mL post-infusion) during this visit.

Additionally, several droplets of capillary blood (approximately 10-20 µL) will be

collected during Visit 1 for screening purposes.

Outcome Measures:

The outcome measures for all three cohorts will be identical as described in the 'Outcome

Measures' section of this protocol