Department of Obstetrics and Gynecology

Literature and Trials: broadly neutralizing antibody (bnAb) administration and protection –Translating the NHP model to human clinical trials

However, how these antibodies are able to reach mucosal sites to elicit protection (i.e. the mechanism) has not been determined.

Currently ongoing human trial• bnAb utilized is VRC01

Pegu et al. Sci Transl Med. 2014 Jul 2;6(243). • 10E8, PG9, and VRC01 (all targeting the CD4 binding site) protected

against high-dose intrarectal SHIV challenge

Klein et al. JVI 2013 May;87(21):11604-11616. • 2F5 protected against vaginal challenge in a dose

dependent manner.

Gautum et al. Nat. Med. 2018 May 24;5(610)• SC administered 3BNC117-LS and 10-1074-LS

protected against low-dose repeated intrarectalSHIV challenges for up to 20 weeks.

Antibody Distribution after IV Instillation (How does it work?)

UC Davis SuperPET18FDG

24 hrs

72 hrs

Ongoing studies suggest it takes at least 1 week to achieve steady state distribution of iv Antibody

Visualizing Antibodies In Vivo

Ecad-FITCCy5-Ab

DAPI

10xEcad-FITC

Cy5-AbDAPI

10xVariable distribution in the mucosal tissue (4 days)

Wait 1 week (?) to get complete systemic distribution of injected antibody Schneider et al. Journal of immunological Methods, 2017

IV injected antibodies take timeTo distribute into the tissue

Visualizing antibodies in vivo at multiple scales (fluorescence)

Fluorescense

Light Sheet Microscopy (3-D)Tissue section imaging

48 hours

Antibodies enter squamous epithelium from below.

• Df kljbqewkb

Positronic Emission Tomography (PET)

• 2 females injected with either:

• Cy5/64Cu VRC01-LS and Cy3 VRC01

• Cy5/64Cu VRC01 and Cy3 VRC01-LS

• PET/CT at 2,4,24,48,72 hrs.

• Necropsy after last PET scan.

• Also have 2 males with PET/CT and 2 males with PET.

Visualizing antibodies in vivo at multiple scales (fluorescence and 64Cu)

Fluorescense Positronic Emission Tomography (PET)

Light Sheet Microscopy (3-D)Tissue section imaging Live animal imaging

48 hours1 hour

Use PET/CT with 3 kinds of probes (so far)Combine PET/CT with fluorescence methods. Powerful enrichment to our fluorescent methods.

1) labeling antibodies (intact) to monitor kinetics and extent of distribution (Fc influence).

2) labeling of SIV/HIV particle to monitor particle distribution.

3) labeling of probe to detect viral envelope. Currently FAB2 anti-SIV envelope antibody. Detection of Viral envelope protein in vivo (envelope expressing cells)

Logistics• CT scan takes 90 seconds

• PET scan takes 15-20 minutes

• Half-life of radioactive probe- longer half-life’s can allow longer kinetics

64Cu 12 hours89Zr 3.3 days

“Hot” monkeys and excrement

Many studies have illustrated that bNAbs have protective properties, but through what mechanism(s)?

• WHOLE ANIMAL LEVEL:• Can we follow Cu64-labeled and Zr89-labeled antibodies

systemically in passive transfer studies in vivo?• Is there a difference in whole animal distribution of VRC01-

Wt and VRC01-LS?

• TISSUE/CELLULAR LEVEL:• Can we follow fluorescently-labeled antibodies systemically

in passive transfer studies in vivo?• Can we identify differences in tissues between VRC01-Wt and

VRC01-LS?• Distribution differences?

• Half-life differences?

Cu64: Differences in VRC01-Wt and VRC01-LS are visible 2 hours post IV injection

VRC01-Wt VRC01-LS

• Both VRC01-Wt and VRC01-LS initially target the liver.

• VRC01-LS accumulation is

more prominent in the transverse

colon.

Cu64: Differences in VRC01-Wt and VRC01-LS are more dramatic 72 hours post IV injection

VRC01-Wt VRC01-LS

• VRC01-Wt persists in the liver and accumulates in the small

intestines.

• VRC01-LS signal is more prominent in the lungs and large

intestines.

• Heterogeneity in the large intestine antibody distribution!

The LS mutation influences anatomical distribution at early time pointsCu64

• VRC01-Wt and VRC01-LS have distinct patterns of early distribution.

• VRC01-LS is found in higher concentrations in the Aorta, Lungs, Heart, Uterus, Colon. HIGHLY VASCULARIZED TISSUES!

• VRC01-Wt initially targets Liver, Small intestine.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

lungs brain thoracicaorta

iliacartery

spine spleen liver kidneys heart GIT ax LN uterus vagina muscle

SU

V

A7X030

2 4 24 48 72

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

lungs brain thoracicaorta

iliacartery

spine spleen liver kidneys heart GIT ax LN uterus vagina muscle

SU

V

97N038

2 4 24 48 72

VRC01-Wt

VRC01-LS

Does the LS mutation influence antibody distribution at the tissue/cellular level??

Zr89• VRC01-Wt and VRC01-LS also have distinct patterns of

early distribution.

• At day 2, VRC01-LS appears at higher concentrations in the Heart.

• By 1 week, both antibodies have a similar distribution.

• Quantification of Zr89 of each organ is still in progress. More animals still to analyze. STILL LOTS TO DO HERE!!

VRC01-LSVRC01-Wt

1wk post injection

d2

d5

d7

Zr89: The 3.3 day half-life of Zr89 allows us to following antibody distribution for almost 3 weeks

d11

d15

d18

VRC01 VRC01VRC01-LS VRC01-LS

Visualizing ADA66004

Day 2

Day 18

PET AT 1 AND 11 DAYS POST

ZR89-PROBE INJECTION

Day 1

Day 1

1

0.0

0.5

1.0

1.5

2.0

SU

V

A14T015 – VRC01 A15X024 – VRC01 LS

3D PET – day 1

Day 1 Day 11

A14T015 – VRC01 A15X024 – VRC01-LS

3D PET – day 11

Day 1 Day 11

A14T015 – VRC01 A15X024 – VRC01-LS

A14T

015 –

VR

C01

A15X

024 –

VR

C01 L

S

Day 1 Day 11

A15X

019 –

VR

C01 L

S

Cu64 - Day 1

• Important notes• Zr and Cu have similar

distribution in these organs

• LS has significantly less signal in the liver and significantly more signal in the heart (especially later)

LIVER AND HEART QUANTIFICATION

A1

4T

015 –

VR

C01

A1

5X

02

4 –

VR

C01 L

S

Day 1 Day 11

A15

X01

9 –

VR

C01 L

S

Cu64 - Day 1

Important notes

Zr and Cu have similar

distribution in these organs

LS has significantly less

signal in the liver and

significantly more signal in

the heart (especially later)

• Previously illustrated that Cy5 and Cy3 labeling of IgG does not significantly alter the antibody (Schneider et al. Journal of immunological

Methods, 2017).

• 10 FEMALE macaques were injected with either a mixture of VRC01-Wt-Cy5/VRC01-LS-Cy3 or VRC01-Wt-Cy3/VRC01-LS-Cy5 (switched).

• Different fluorescent tags, switched, will allow us to see the antibody differences between each animal regardless of tissue background.

• Macaques were necropsied at various time points (24hr, 48hr, 72hr, 1wk, and 2wk).

• Tissues were frozen down in optimal cutting temperature (OCT).

From these animals, we can illustrate that VRC01-LS persists longer in macaque tissues than VRC01-Wt. HOMEOSTASIS MECHANISM?

(TISSUE/CELLULAR LEVEL)Can we identify differences in tissues between VRC01-Wt and VRC01-LS in

passive transfer studies?

LSWT

LS WT

Paraffin embedded tissues from 2 different animals with the fluorescent tags switched.

Can we visualize LS specific effects in endothelial cells?

• LS mutation increases affinity to FcRn.

• Do we see LS specific differences in endothelial cells?

2 WeeksYES! We can visualize (Deconvolution Microscopy) these differences of VRC01-LS in the

brain.

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of

mice.(Borvak et al. Int. Immunol., 1998)

- Demonstrated that FcRn is expressed in functionally active form in endothelial cells, and are a possible site at which serum IgG

homeostasis is maintained

Recycling EndosomeSalvaged VRC01-LSreturned to surface

VRC01-WtVRC01-LS

Capillary in macaque brain

24hrs post injection

1) VRC01-Wt (green) and VRC01-LS (red) endocytosed together (yellow).

2) VRC01-Wt and VRC01-LS sorted in sorting/recycling endosome. LS will preferentially bind to FcRn.

3) VRC01-LS returned to circulation or distributed to tissue. VRC01 is degraded.

Visualization of a mechanism of LS function (FcRn in action?)Will VRC01-LS be separated from VRC01-Wt

in the recycling endosome?

To advance this work, a “good” Antibody to FcRn is required

• Rozanolixizumab – Therapeutic Ab to IgG binding site of FcRn

• “Underground Ab World”

• Success!

FcRn is expressed in capillaries

We find VRC01-LS in the same capillaries!

60 Macaque brain 24hrs post injection – single antibody (VRC01-LS) animal

72 hours mixture of VRC01 and VRC01-LS

Concentration in Capillaries = Less Ab in Tissue?

VRC01

VRC01-LSVRC01

VRC01-LS

In capillaries, we can

visualize VRC01-LS,

and not VRC01-Wt,

preferentially trafficking with FcRn

Capillary in Macaque brain 24hrs post injection

VRC01VRC01-LSDAPI

VRC01VRC01-LSFcRn

VRC01 VRC01-LS FcRn

VRC01

VRC01-LS

FcRn

20

CD68FcRnDAPI

CD68

FcRn

CD68 FcRn

In rectal mucosal tissues, FcRn is found associated with macrophages!

• FcRn activity in macrophages is essential for regulating IgG homeostasis

• FcRn is functionally expressed in immune cells

However, when staining for FcRn, it is difficult to determine if FcRn is expressed in mucus-producing simple columnar tissues!

• 2 macaques IV-injected with fluorescently-tagged anti-FcRn (8mg/kg).• Necropsy 72hours later.

NON-SPECIFIC BINDING IS A PROBLEM!

DOI: 10.1371/journal.ppat.10037

76

FcRnECADDAPI

FcRnAutofluorescenceDAPI

Endocervix

In mucosal tissues, IV-administered, fluorescently-tagged FcRn is found only in the lamina propria, not in the columnar epithelia!

FcRnECADDAPI

FcRnAutofluorescenceDAPI

Rectum

DOI: 10.1038/nature13612

FcRnECADDAPI

FcRnAutofluorescenceDAPI

Uterus

DOI: 10.1073/pnas.1012861108

40

Human Vagina(IF)

FcRnDAPI

In squamous mucosal tissues, FcRn is also found in the lamina propria and NOT the epithelium!

VRC01-LSECADDAPI

Macaque Vagina

If there is no FcRn in the vaginal epithelium, how is antibody getting to the luminal surface?

DOI: 10.1371/journal.ppat.1003776

DOI: 10.1073/pnas.1012861108

FcRnDAPI Macaque Vagina

(IV injected)

Modulating effector function through FcgR binding

Name Mutation Comment Reference

N297A N297A Reduced interactions with all FcγRsLeabman et al, Mabs (2013) 896-

903

LALA L234A/L235AReduced binding to FcγR1a,

FcγRIIa, FcγRIIIa and FcγRIIIb

Hessel et al, Nature (2007) 449,

101-104

K322A K322A Reduced C1q and C3 bindingHessel et al, Nature (2007) 449,

101-104

LALA/K322A L234A/L235A/K322ACombination of LALA and KA

mutations

FES L234F/L235E/P331S Reduced FcγR and C1q bindingOganesyan et al, Acta Crystallogr D

Biol Crystallogr (2008) 64 (700-704)

DEL S239D/I332E/A330LReduced FcγRIIb and enhanced

FcγRIIIa binding to improve ADCC

Lazar et al, PNAS (2006) 103, 4005-

4010

DEA S239D/I332E/G236AImproved FcγRIIa/FcγRIIb binding

ratio to improve ADCC

Richards et al, Mol Cancer Ther

(2008) 7, 2517-27

FT H268F/S324TIncreased C1q binding without

affecting FcγRMoore et al, mAbs (2010) 181-189

FTDEH268F/S324T/S239D/I332

EIncreased binding to C1q and FcγR Moore et al, mAbs (2010) 181-189

Slide from Koup, 2019

LALA(L234A/L235A)

DEL(S239D/A330L/I332E)

FcgR

C1q

CH2

CH3

239332

330

332

330

DEL - Reduced FcγRIIb and enhanced FcγRIIIa

binding to improve ADCC

DEL Mutation (VRC07-523-LS-DEL Alters Ab Cellular Interactions at Mucosal Sites (Buccal)

DEL Mutation (VRC07-523-LS-DEL and LALA (VRC07-523-LS-LALA ) Alters Localization (colon)

VRC07-523-LS-LALA accumulates in a subset of peripheral Goblet cells

WTF cells?????

The LALA mutation in the context of VRC07-523-LS-LALA leads to an unexpected localization within a subset of luminal proximal goblet cells in the colon. We have never seen this before. Analysis to identify additional acquired localizations of the VRC07-523-LS-LALA is ongoing

VRC07-523-LS-DELVRC07-523-LS-LALA

VRC01-LS vs. VRC01-523-LS72 hours.

Conclusions The distribution of IV injected antibodies is complicated!

WHOLE ANIMAL LEVEL:• Antibodies are distributed to different anatomical sites by distinct mechanisms.• Cu64: At early time points, the distribution of VRC01-Wt and VRC01-LS differs with LS

distribution found mainly in highly vascularized organs.• Zr89: By one week, VRC01-WT and VRC01-LS have a similar anatomical distribution.

CELLULAR/TISSUE LEVEL:• During IgG homeostasis in endothelial cells, VRC01-Wt is degraded while VRC01-LS is

able to bind to FcRn and is; therefore, returned to circulation (or delivered into tissues), stockpiled in endothelial cells????.

• In mucosal tissues, FcRn is found below the epithelium and not in the epithelium itself.

• We are just beginning to appreciate the complexities here. Clearly more work is needed.

ACKNOWLEDGEMENTS CollaboratorsRuth Ruprecht

Sandeep GuptaPeter FoxSid KumarapperumaBeth GoinsSiqi GongFrancois VillingerPhil SantangeloMariluz AraíngaDaryll Vanover

Rosemary BastianDanijela MaricJoão MamedeKatarina KotnikRoslyn TaylorEd AllenYanille ScottJoe Griffin

Koree AhnShoaib MuhammadLisette CorbinPat MaddenIsabelle ClercGianguido CianciDivya Thakkar

WE ARE LOOKING FOR POST-DOCS!

Post-doctoral positions available to use PET/CT to define the paradigm

John MascolaAmar PeguCasandra AlmasriRick KoupRon VeazeyGenoveffa Franchini