Correction of Underlying Lymphoid Glucose Utilization Defects with the BCG Microorganism:
Implications for Type 1 Diabetes through Quantitative Method Development
G. Shpilsky, Y. Song, W.M. Kühtreiber, T. Luck, M. Weil & D.L. Faustman
Massachusetts General Hospital and Harvard Medical, Boston, MA, USA

Introduction

We investigated baseline defects in aerobic glycolysis in T1Dversus NDC and evaluated the impact of BCG on glucose
utilization in vitro and in vivo through a clinical studyResults•

T1D lymphocytes had defective baseline 2-NBDG uptake(indicating underactive aerobic glycolysis) and high oxygenconsumption (indicating overactive oxidative phosphorylation) thatwas corrected after 24-hr exposure to BCG in vitro (Fig. 1)o•••Control

Increased sugar uptake could also be observed in NDC subjects
after BCG exposure, but the effect was more dramatic for T1D

In T1D, the severity of the glucose utilization defect was mostpronounced with age of onset (AOO) < 21 years compared toAOO > 21 years (Fig. 1)
AOO influenced BCG’s ability to accelerate glucose uptake inT1D both in culture and in an open-label study in the clinic (Fig 2)
Prior exposure to BCG at birth resulted in life-long accelerated
glucose uptake in NDC (Fig. 3), which was also observed:oo

In NDC with prior BCG treatment of bladder cancer
In T1D with latent tuberculosis infection

5000

***

3000
2000
1000

T1D––T1D–+0

NDC

Fig 1. Glucose transport by T1D and NDC human primary monocytes as measured by 2-NBDG flow cytometry. All subjects
were without childhood exposure to BCG. (A) The 2-NBDG assay for 4 hrs of labelled sugar quantifies the rate of glucose
utilization by monocytes and flow cytometry. (B,C) Untreated baseline relative glucose uptake was significantly different for NDC(n=23) and T1D (n=43) patients (p<0.0043; 2-tailed, unpaired T-test). BCG increased relative glucose uptake for monocytes ofNDC and T1D patients. 2-tailed, unpaired T-test revealed a significant difference between NDC and T1D patients (p=0.045).
Untreated baseline and BCG treated baseline relative glucose uptake for T1D show a significant difference between untreated andBCG treated samples (p=1.837 x 10-5; 2-tailed, unpaired T-test). (D) Untreated baseline subtracted from BCG treated baseline
relative glucose uptake for T1D patients separated by AOO < 20 and > 20 y of age compared to NDC. 1-tailed, unpaired T-test
revealed a significant difference between untreated vs BCG-treated samples (p=0.02).

DiseaseIn vivo BCG
In vitro BCG

CBCG decreases HbA1c in patients with T1D, but HbA1c
effect is related to age of onset (AOO)
A50B

T1D
AOO < 211520T1D
AOO > 211-5-5-10-10Yearsp=0.02

CYearsp=0.91

2100246Years

T1D–+Countryat Birth

BCG Strain

1

Newborn Vaccination

India

India

25

Birth

2

Bladder Cancer

USA

TICE

71

Adult

3

Newborn Vaccination

MonacoSanofi
Pasteur

42

Birth

4

Newborn Vaccination

Hungary

Danish

39

Birth

5

Newborn Vaccination

India

India

25

Birth

6

Newborn Vaccination

UkraineBulgaria

49

Birth

7

Bladder Cancer

USATICE

71

Adult

8

Newborn Vaccination

Hungary

Danish67

BirthCurrent
AgeDosingAgeWe conclude that NDC monocytes have different levels of basal lymphoidsugar utilization compared to T1DoT1D-Reference
T1D-Open Label

T1D––NDC–+Fig. 3. (A) Glucose uptake in monocytesof NDC patients (n=23) following 24-h
incubation with or without in vitro BCG
treatment. Samples marked in green arefrom individuals with previous BCG
vaccinations in childhood or who received
BCG as treatment for bladder cancer.
Samples marked in black show the effectof BCG treatment in vitro on glucoseuptake with 4 hrs of labelled 2-NBDG
compound. In NDC, sugar update isaccelerated. The same experimental
conditions were applied to NDC who had
BCG at birth or had received high dose
BCG for bladder therapy. In vivo therapy
with BCG augmented sugar utilization.
(B) Glucose uptake in monocytes of T1D
patients (n=43) following 24-h incubation
with or without in vitro BCG treatment.
The sample marked in green represents
an individual with T1D with remarkablediabetes management (63-y history ofexcellent HbA1c control; AOO 12 y;current age 75 y) and a lung mass
suspicious for latent M. tuberculosis
infection (signs of tuberculosis treated 20years earlier on chest X-ray). Samples
shown in red show the in vitro responseof BCG exposure on monocytes followedby 4 hrs of measured 2-NBDG uptake.
(C) Summary of past BCG exposures,
strain of BCG and current ages ofsubjects in which BCG appears to keep
the cells under excellent aerobic
conditions. The long-lasting and near lifetime effect of BCG treatment (vaccine orbladder infusion) may permanently modify
monocyte glycolysis for accelerated
aerobic glycolysis.

Conclusions

T1D-BCG
T1D-Placebo

Fig. 2. (A) HbA1c % change from baseline in T1D patients receiving open-label BCG treatment with AOO < 21 y (n=5) compared
to a T1D reference population not receiving BCG treatment (n=62) for 2 years. (B) HbA1c % change from baseline in T1D patients
receiving open-label BCG treatment with AOO > 21 y (n=8) compared to a T1D reference population not receiving BCG treatment(n=62). (C) HbA1c % change from baseline in T1D patients receiving open-label BCG treatment (n=5) (red line) compared topreviously published data of T1D from an earlier double-blinded, placebo-controlled study, verifying in the open-label trial thetrends and effects based on juvenile versus older AOO in T1D subjects.

NDC––

Subject Indication

8•

-10

-20

5000

RL

20
T1DBCG

10000

CT

20
NDC
–+O>

O<Disease NDC
In vivo BCG–
In vitro BCG–AO

0B*15000*4000

0We sought to better understand lymphoid defects in sugarutilization in T1D and present findings in T1D and non-diabeticcontrols (NDC)

Methods•***10000

A5000

2-NBDG (MFI)

Vaccinated subjects had lymphoid restoration of glucose
metabolism through a shift from oxidative phosphorylation to aerobic
glycolysis, correcting defective glucose metabolism (Kuhtreiber et al.
Nature Vaccines 2018 & Cell Trends Endocrinol Metab 2019)
This occurred via the BCG organism epigenetically reprogramming
glucose utilization pathways (Kuhtreiber et al. Cell iScience 2019)

BCG increases relative glucose uptake in T1D and NDC human primarymonocytes; Past BCG exposure results in life-long augmentation

D**AO•

C150002-NBDG (MFI)BCG Stimulated-UntreatedoB

HbA1c % changeoA

2-NB DG (M F I)

Human clinical trial data, including 8-year follow up of HbA1c
after two BCG doses, shows long lasting blood sugar control intype 1 diabetes (T1D)

HbA1c % change•

BCG corrects an underlying defect in glucose utilization in T1D
monocytes; More severe defect in early age of onset (AOO)

T1D lymphocytes have lower levels of basal lymphoid sugar utilization compared toNDC, but in culture this is corrected with BCG•AOO of T1D influences the severity of glucose utilization defects in vitro and invivo and influences in vivo correction of blood sugars with BCG vaccination•Prior BCG vaccinations at birth appear to “permanently” reset monocyte
glucose utilization, thus influencing sugar utilization patterns
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Contact: dfaustman@mgh.harvard.edu

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Optimizing TNFR2 Antagonism for Cancer Immunotherapy:
Antibody Structure & TNFR2 Density on Target Cells Drive Tumor Microenvironment Specificity
M. Yang, K. Case, W.M. Kühtreiber, D.L. Faustman
Massachusetts General Hospital and Harvard Medical, Boston, MA, USAIntroduction••

Results
Use of an IgG2 antibody reduced the dependency
on Fc receptor function

Most current immunotherapies lack regulatory T cell (Treg)
specificity for tumor-residing cells or tumor-specific killing forbroadly expressed oncogenes
Tumor necrosis factor receptor 2 (TNFR2) antibody antagonism
may be an attractive cancer immunotherapy because it has showntumor microenvironment specificityoNatural IgG2 antibodies invivo and in vitro can make2-5 different isoforms. We
investigated which (or if all)isoforms have antagonisticactivity. We performed SCX
fractionation to physicallyseparate the antibodyisoforms. These data showthat only one type of
shuffled IgG2 isoform of a
TNFR2 antagonist
maintained the desired Tregand tumor activity. This
isoform is isoform F5.

Human TNFR2 is massively overexpressed on tumor-residing Tregs,
under-expressed on tumor residing T effectors (Teffs) and founddirectly on some cancer cells (Yang et al. J Leukoc Biol. 2020)

Methods•••

We screened 791 human tumor cell lines from diverse cancer
tissues, finding frequent and aberrant TNFR2 expression (below,left) and examined the density of TNFR2 on Tregs, Teff and cancer
cells in cutaneous T cell lymphoma vs normal controls (below, right)
Using 3 cell-based tumor microenvironment assays, we tested anovel human-directed TNFR2 antagonist placed on different humanisoform frameworks
Using mutagenesis techniques, we tested different mutations to ournovel TNFR2 antagonist on human chimeric structures to optimize
function

DMutated hinge variants of IgG2 produce single and stableisoforms with different functional activity in cell-based tumor
microenvironment assays

Chimeric human IgG2 antibodies were constructed with hinge mutations at (A) C232S andC233S, (B) C232C only, and (C) C233S only. (A) Human IgG2 isoform with hingestabilization mutants C232S, C233S showed a single band on a nonreducing gel andrestored Treg killing, Teff proliferation and SW480 tumor cell killing. (B-C) Human IgG2isoform with hinge stabilization mutant C232S or C233S showed a single band on anonreducing gel, but no Treg killing, good Teff activity, and sluggish SW480 tumor killing. Arepresentative experiment is shown for the Treg and Teff experiments, performed >5 times
on different normal blood donors. For the SW480 cells, the error bars represent 8 separateexperiments. Data are mean ± SEM.

Conclusions••

These findings suggest that the ideal TNFR2 antagonists are thehuman IgG2 isoform, have hinge stabilization and have wider
separation of antibody arms to bind to newly synthesized TNFR2 on
rapidly growing tumor cells or Tregs specific to the tumor
microenvironment
When bound to TNFR2, antagonistic antibodies with thesecharacteristics form a non-signaling cell surface dimer that functions
as an effective immunotherapy with high tumor microenvironment
specificity and desired modulations
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Contact: dfaustman@mgh.harvard.edu

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