#562

Antibodies to SIRPa enhance innate and adaptive immune responses toto promote anti-tumor activity

Tracy C. Kuo, Amy Chen, Ons Harrabi, Steven E. Kauder, Emma R.B. Sangalang, Laura Doyle, Sangeetha Bollini, Bora Han, Janet Sim, Jaume Pons, and Hong I. Wan
ALX Oncology, Burlingame, CA
Background & Objective• H21 binds similarly to SIRPg on primary human CD4 and CD8 T-cells (A).
• CD4+ and CD8+ T-cell proliferation in a mixed lymphocyte reaction with allogeneic monocyte derived• Clone 21 potentiates anti-PD-1 tumor inhibition in MC38 syngeneic model

2000

10000.01

1100

10000

EC50 6.2 nM

EC50 0.75 nM
0.01

anti-Sirpa Log[nM]

1100• H21 induces phagocytosis by SIRPa homozygous and heterozygous v1 and v2 macrophages andpotentiates the efficacy of anti-tumor and anti-PD-1 antibodies in xenograft and syngeneic models,
respectively.

10000

00.0001

0.01

1100

specific anti-SIRPa antibody (HEF) fails to induce phagocytosis by v1/v2 and v2/v2 macrophages.

C1510
500.0010.010.1

110

anti-Sirpα Log[nM]1001000

504030201000.001

0.010.1

110anti-Sirpα Log[nM]1001000

00.010.1

1101001000

h21 Log[nM]

H21 enhances anti-tumor activity in combination with rituximab in theRaji xenograft model

1αP
D

1αP
D+
2121% of CD4% of TAMs

1001.01αPD

121++αPD

21PBαPD

S-11αP
D

21S

21+0.0PB

1αP
D

121αP
DPB+
2121S

1αP
D1αP
D

2121+PBαP
DS-11αP
D21SPB+% of CD41.5200.50S0.002121242.0200.5% of CD8% of CD451αPDSPB1Median Fluorescent
Intensity (MFI)% of CD81αPDαPDum

H

PB

4Tumor:IFNγ+ ofg+ CD4IFN
TCD4cells+ TAMs
Tumor:of TAMS
iNOSiNOS
30

61000

400+ T
IFNγ
ofcells
CD4g+ CD4
IFNSpleen:
82000

60

2000PB

61.0

402053000

Median Fluorescent
Intensity (MFI)

8H21 was well tolerated in cynomolgus monkeys with favorable PK andextended receptor occupancy• H21 was administered on days 1 and 8 via intravenous injections at 10 and 30 mg/kg• No test article related adverse signs were observed in clinical evaluations, hematology and serum••

chemistry.
PK parameters show extended half-life expected of antibody and full target occupancy was observed
on monocytes.
No test article related changes were observed in B-cells, NK cells, T cells and monocytes for treatedanimals.

PKPK
1000

2623 (PBS)

2629 (10mpk)

2631 (30mpk)

2624 (PBS)2630 (10mpk)

2632 (30mpk)

media only
100

20

rituximab 2/10 TF

1510
500.001

0.01

0.1

110

anti-Sirpα Log[nM]

PBS

2000

v2/v2 SIRPαmacrophages

Tumor volume(mm3± SEM)

20v1/v2 SIRPαmacrophages% Phagocytosis(%CFSE+ of macrophages)

Barclay AN, Van den Berg TK (2014) Annu Rev Immunol 32:25–50.
Lakhani N, et al. (2018) SITC, Abstract P335.
Sim J, et al. (2019) Keystone Symposia, Abstract Poster 3022
Kauder SE, et al. (2018) PLoS One 13(8):e0201832.

v1/v1 SIRPαmacrophages% Phagocytosis(%CFSE+ of macrophages)

1.
2.
3.
4.

% Phagocytosis(%CFSE+ of macrophages)

References:IgG control

1021nti-62CCC
1.5Raji• PK/PD profile of h21 in cynomolgus monkey showed typical antibody PK and complete SIRPαoccupancy. H21 was well tolerated with no adverse signs observed in clinical evaluations,hematology and serum chemistry

cetuximab

2000

8060

15- DC-DC
CD86CD86
MFImedianof CD8
Spleen:of CD8

free mice in combination and rituximab only groups, respectively.

HEF2500

Tumor: Granzyme
Granzyme
B CD8B+ ofT CD8cells
10080203000

Dendritic Cells

Tumor:TAMs
TAMs of CD4525

1hIgG1 inactive control+ ofSpleen:
IFNγg+ CD8IFN
T CD8cells

3500

αPD

hIgG1 control• Combination of h21 with rituximab induced tumor regression. Treatment led to 6/10 and 2/10 tumor• Clone 21 in combination with anti-PD-1 induces DC activation, increases M1 tumor associated* p < 0.05** p < 0.005*** p < 0.0005

25Tumor+DC+ DC
Spleen:of CD8CD86CD86
MFImedianof CD8210.01 ug/mL Cetuximab

10000• H21 induces phagocytosis by homozygous v1 and v2 and heterozygous v1/v2 macrophages while allele

h2120SpleenSH21-IgG1

anti-Sirpa Log[nM]anti-Sirpa Log[nM]

H21 binds v1 and v2 SIRPa alleles and induces phagocytosis

macrophages (TAMs) and T-cell effector function thus bridging innate and adaptive immunity inMC38 tumor-bearing mice.

•1500• Anti-SIRPα clone 21 was selected for humanization and displays high affinity binding to both humanSIRPα alleles v1 and v2, cynomolgus monkey and mouse SIRPα monocytes.

15+EC50 5.2 nM

1000

00.0001

101001000

1500

2121+ rituximab 6/10 TF

1000

***

****

500
051015Days post implantation

20*** p < 0.0005**** p < 0.00005

21: 10 mg/kg, 4q3d
rituximab: 3 mg/kg, 4q3d

MonocyteOccupancy
Occupancy
Monocyte
SIRPa occupancy, percent(normalized to predose)

1000

5IFNg expression in splenic T-cells stimulated ex vivo with PMA/ionomycin.
Increased inducible nitric oxide synthase (iNOS) expressing TAMs (M1 phenotype) and T-cell function
in group treated with 21 in combination with anti-PD-1.

Anti-SIRPα
Antibody Concentration µg/mL

2000

H21-IgG4

Media onlyMouse(C57BL/6)

3000

EC50 0.62 nM

H21-IgG1 inactive

H21-hIgG1 inactive
isotype control

KWAR

3000

2000

B.

H21-hIgG1 inactive

HEF

4000

EC50 0.55 nM(anti-S(a•media onlyCynomolgus Monkey

EC50 0.32 nM

21: 10 mg/kg, 2q3dAnti-PD-1: 10 mg/kg, 1 dose500• Tissues were harvested two days post last dose.
• 21 alone and in combination with anti-PD-1 induced splenic dendritic cell activation and increasedIgG1 and IgG4 diminish the effect (A).
H21 with effector function depletes dendritic cells (HLADR+Lin-) in PBMCs while h21 with inactive Fcfunction has no effect (B).
H21 does not affect cell counts for B-cells, T-cells, NK cells and monocytes.

H21-hIgG4

Human (v1/v2)

D47an)
izehIdgG
21Conmtr
Ig
olGC
ontrolDC:Tonly
TonlyPγ)IR00

h21 Log[nM]0•

21 + anti-PD-1** ***

10000H21-hIgG1Isotype controlh21*00V2cynomolgus mouse<1.00E-12
3.61E-12
4.60E-10
4.60E-12
3.89E-12
9.25E-09
NBNBNB
9.57E-09
5.25E-09
NB21

anti-PD-1
1500

Days Post Tumor Implantation

Fold over Media only

V1
<1.00E-12
2.06E-12
1.94E-09
5.23E-10

80000

21

2000

40000

A.
KD (M)
21h21HEF
KWAR

Tumor volume (mm3),mean ± SEM

Cell Count200

CD8120000

PBS• H21 with reduced effector function potentiates cetuximab induced phagocytosis of DLD-1 cells, while

antibodies.

00.0001

CD8400

CD4Reduced effector function is optimal for potentiating phagocytosis andminimizing Fc-mediated dendritic cell depletion• H21 binds SIRPa across species and with similar affinity as parent clone 21.
• Similar binding profile of h21 and 21 on primary human, cynomolgus and mouse monocytes.
• On v1/v2 human monocytes, h21 binds with the lowest EC50 as compared to known anti-SIRPa

3000

CD410Block
Block
Block
Kick-offNon-block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block
Non-Block02.67E-10
3.42E-10
5.39E-10
1.10E-10<1.00E-12 <1.00E-12
B
B
2.39E-08
4.35E-09
8.36E-08
1.63E-09
NB
B
8.99E-09
B
2.02E-09
1.71E-08
7.47E-10
7.07E-08
1.89E-09
2.06E-10
2.84E-09
2.63E-09
3.97E-09
3.00E-09
9.47E-10
5.36E-08
6.36E-11
B
2.71E-08
1.06E-08

10NB
NB
1.05E-09
NB
3.50E-10
1.16E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

10

NB
NB
4.60E-10
NB
1.27E-08
3.65E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

1NB
NB
5.47E-10NB
5.54E-10
1.63E-09
NB
NB
NB
NB
NB
NB
NB
NB
NB
NB

160000600

CD47 Blocking

Mixed Lymphocyte Reaction

H21-hIgG1

coverage was identified and will be useful tools for interrogating SIRP biology and the CD47-SIRPαcheckpoint.

1.12E-10
9.69E-11
3.61E-12
2.41E-09
2.15E-09
2.29E-09
4.46E-08
3.90E-08
4.49E-08
1.62E-09
7.64E-09
6.35E-09
1.26E-08
9.11E-10
5.99E-11NB

SIRPb1

H21 binds human, monkey and mouse SIRPaMedian FluorescentIntensity (MFI)• A panel of anti-SIRPα antibodies with diverse binding profiles, SIRP reactivity and broad epitope

BALB/c• All antibodies are screened as full IgG1 except 173, 209, 213, 123, 149, 161, 162, 194, 218 are screened as scFv-Fc• NB = no binding; B = binding confirmed and exact KD value was not determined• W=wt, H=OmniChicken, S=SynVH chickens

4000

Concluding remarks

BL61

1.83E-10
6.82E-11
1.51E-10
2.90E-11<1.00E-12 <1.00E-12
4,26E-10
1.86E-09
4.58E-10
1.63E-09
1.62E-10
7.67E-11
9.37E-10
9.28E-09
1.71E-10
5.01E-09
6.05E-09
1.69E-09
6.05E-10NB
8.73E-10
2.38E-10
1.03E-09
1.27E-10
4.50E-10
1.57E-08
4.97E-10
NB
1.23E-10
2.76E-10
6.63E-11
1.34E-10

NOD

0.

H
H
S
H
H
W
H
S
S
H
H
H
H
H
S
W

SIRPg

Cyno

01

119
135
21115
136
3173
209
213
123
149
161
162
194
218
45

V2

0.

1
1
12
3
4
44
4
5555556V1

1Source

MC38
2500

B.

Human PBMC

00

AntibodyA.

01

Bindendritic cells are inhibited by specific anti- SIRPg and anti-CD47 antibodies but not by h21 (B).

00

•

0.

nanomolar to picomolar (KD,).
A select number of clones (21, 119, 135, 136) bind similarly to human SIRPα alleles and cynomolgus
SIRPα; in addition, clones 21 and 136 cross-react with mouse SIRPα alleles, enabling preclinical mousestudies.

22kinetic mode and flowed over immobilized anti-SIRPα antibodies. Epitope binning was carried out
using a classical sandwich approach3.
• Humanization. Chicken variable regions of the light chain were grafted onto human lambda lightchain IGVL3 frameworks.
• Cell binding on primary human, monkey and mouse monocytes. Parent and humanized 21 werelabeled with Alexa Fluor 647 and 1:4 titration of the antibodies starting at 500nM were incubatedwith human, cynomolgus PBMCs and mouse splenocytes. Monocytes in human and cynomolgus
PBMCs were identified by CD14+CD16- and in mouse splenocytes by LY6C+MHCII-.
• Phagocytosis. In vitro monocyte-derived macrophages were polarized using M-CSF or human serum
as previously described4.
• Mixed Lymphocytes Reaction. Mature DCs cultured in CD47-Fc coated wells were generated frompurified monocytes treated with IL-4 and GM-CSF and stimulated with LPS/IFNg . DCs and T cells at
a 1:5 ratio were incubated along with 10µg/mL of antibody.
• PBMC depletion. PBMCs were incubated with 10µg/mL of antibody. Cell viability was assessed witha cocktail of cell-specific antibodies two days post treatment.
• Tumor models and immunophenotyping. Raji and MC38 cells were implanted in NOD SCID and
C57BL/6 mice. Harvesting and processing of tumors and spleen from MC38 tumor bearing mice
were performed as previously described4.
• Tolerability, Pharmacokinetics and Pharmacodynamics study in cynomolgus monkeys. Antibodies
were administered intravenously on days 1 and 8 at 10 or 30 mg/kg. Serum concentrations weredetermined using SIRPa capture enzyme linked immunosorbent assay. Receptor occupancy wasmeasured using a flow cytometry assay with labeled H21.

• A diverse panel of antibodies bind to human SIRPα v1 and v2 with high affinity ranging from lowLSB• Surface Plasmon Resonance (SPR). For KD determination, SIRP analytes were injected in a “one-shot”21 enhances anti-tumor activity in combination with anti-PD-1 in MC38
syngeneic tumor model

0.

Methods

H21 binds SIRPg but does not impact T-cell proliferation in mixed
lymphocyte reaction

Median Fluorescent
Intensity (MFI)

Objectives of our study:• Characterize anti-SIRPα clone 21 for binding to human, mouse and cynomolgus monkey SIRPα.
• Phagocytosis of target cells by prevalent alleles of human SIRPα (v1, v2) macrophages.
• Identify optimal IgG isotype for humanized 21 (h21).
• Evaluate clone 21 for anti-tumor activity in both xenograft and syngeneic tumor models andelucidate the mechanism driving efficacy.
• Evaluate h21 in cynomolgus monkeys for pharmacokinetics (PK), pharmacodynamics (PD) andtolerability

Immunization in chicken produced diverse high affinity anti-SIRPα
antibodies that can potentiate macrophage phagocytosis% Phagocytosis(%CFSE+ of macrophages)

Signal-regulatory protein α (SIRPα) is an immune checkpoint receptor expressed primarily on myeloid
cells and neurons1. SIRPα suppresses innate immunity upon interaction with its ligand, CD47. Targeting
the CD47-SIRPα pathway represents a novel therapeutic approach to enhance anti-cancer immunity.
Ongoing clinical trials to inhibit this pathway through targeting CD47 have shown promising results inreducing tumor burden2. Unlike CD47 which is expressed ubiquitously, SIRPα expression is more
restricted. Therefore, targeting SIRPα may result in differential safety and efficacy profiles versus thatof CD47-targeted therapies.

Results

1010.1

0.01

084

168

252

Hours

336

420

504

100
8060
40200-20-40-60-80084

168

252

Hours336

420

504