ANGN BIOM : Lymph Node Targeted AMP-peptide Vaccines Generate Functional T cell Immunity Against Mutant p53 and BRAF (SITC – Scientific Poster)

Presented at the SITC 38th Annual Meeting, 3-5 November 2023, San Diego, CA, USA, and online	Abstract 1145

Lymph Node Targeted AMP-peptide Vaccines Generate Functional

T cell Immunity Against Mutant p53 and BRAF

Martin P. Steinbuck1, Xavier Cabana-Puig1, Erica Palmer1, Mimi M. Jung1, Thomas Williams1, Kristen Osaer1, Jeff Zhang1,

Christopher M. Haqq1, and Peter C. DeMuth1

1 Elicio Therapeutics, Inc. 451 D St., Ste 501, Boston, MA 02210

Clinical Relevance: p53 and BRAF Mutations in Cancer

Repeat-dose Immunization Strategy		ELI-008 Is Designed to Target >30% of p53 Mutations

Mutant p53

Nearly 60% of human tumors exhibit dysregulated p53[1]

including mutations, copy number alterations, upstream deregulation

~50% of those tumors have missense mutations in p53[2,3]

Annual incidence: 5.2M patients worldwide and 700K patients in the US

Prevalent in nearly all tumor types; sign of advanced disease Poor clinical prognosis[4]

p53 Mutations by Cancer Type

Estimated Worldwide Annual Incidence[2,3]

Brain

Mutant BRAF

Mutations are concentrated in one hotspot[5-11]

Up to 97% of BRAF mutations occur at the V600 location

High prevalence in melanoma and thyroid tumors[10,11]

Overall, BRAF mutations are present in 8% of solid tumors Representing approx. 1 million cases globally each year

BRAF Mutation Hotspots

Melanoma[11]Thyroid[5]

5%

8%

V600E	V600E
V600K
V600X	Other

Preclinical Murine Doses:

Mutant

% Patients

AMP-CpG

AMP-Peptide

1

R248W

3.53%

Day

Day

Day

Day

Day

Day

ELI-007

10 nmol

+

20 nmol

2

R248Q

4.37%

Schema

C57BL/6

-ELI008

Adjuvant [TLR9 Agonist]

3

R175H

5.60%

1

7

14

21

28

35

ELI-008

5 nmol

+

1.25 nmol

Potential candidates: 12 of the most

4

R273H

3.95%

ICS, Luminex

AMP-CpG 7909

*Dose

Preclinical Assays:

5

R273C

3.31%

~30% of all

Antigen [30mer Peptides]

6

R282W

2.83%

patients with

ELISpot, FluoroSpot

AMP-p53 Mutant Peptides

7

G245S

2.11%

Assay

Spleen

p53 mutations

8

R249S

2.04%

Blood

ICS

frequently occurring p53 mutations[21].

9

Y220C

1.83%

*Control groups were immunized with equivalent amounts

10

C135Y

0.43%

Lung

ELISpot, ICS

of unmodified "soluble (SOL)" CpG and antigen peptide.

11

R158H

0.52%

12

H214R

0.39%

Ovary	Total Cancer Incidence
Melanoma
Kidney
Leukemia	~30%
Pancreas
Non-Hodgkin
Bladder	~60%
Thyroid
Esophagus	p53 Mutation
Cervix
Liver	p53 Dysregulation
Stomach
Prostate
Colorectum
Lung
Breast
1	2

Incidence of most common cancers

(in millions)

84%	Other	95%

Most Frequently BRAF-Mutated Cancer Types[10]

100%	Hairy Cell
	Leukemia[7]
	Thyroid[6]

60%

11%

Melanoma[8]Colorectal[9]

54%

ELI-007 is Designed to Address ~95% of BRAF Mutations

007	Adjuvant [TLR9 Agonist]		Mutant	% Patients
1	V600E	~90%
AMP-CpG 7909
	2	V600K	~5%
ELI-	Antigen [29mer Peptides]
AMP-BRAF V600E		~95% of all patients
	AMP-BRAF V600K		with BRAF mutations

ELI-007 Generates Strong Immune Responses to V600E and V600K

AMP-p53 R248W Produces Strong T Cell Responses in Mice

The 10mer, 18mer, and 30mer AMP-peptides are centered around the same R248W mutation.

		T Cell IFNγ Response: Spleen Day 21		T Cell IFNγ Response: Spleen Day 35
		Stim: p53 R248W OLPs		Stim: p53 R248W OLPs
splenocytes	12000				splenocytes	25000
7000
				15000
2000	>9x	>50x	>28x	>26x	>530x	>10x
2000
6				6	10000
/ 1x10					/ 1x10
1500
1000
SFC				SFC	5000
500
IFNγ				IFNγ
0				0

Therapeutic Vaccination: Benefits of Targeting Mutated p53 & BRAF

Peptide

SOL AMP SOL AMP SOL AMP

Peptide

SOL AMP SOL AMP SOL AMP

Truncal Mutation

Driver Mutation

High Prevalence

Public Neoantigen

Proven Clinical MOA

Mutant p53

Mutant BRAF

These mutations are present early on in tumor progression and shared among all progeny as they apply positive selection for survival in the cancer cell population and allow for clonal expansion[12].

Signaling is required for tumor survival and growth, making loss of

these mutations unlikely, thus preventing immune escape.

	Major driver in [10]
Occurs in nearly all types of	100% of hairy cell leukemia
tumors and is involved in	60% of thyroid cancer
~30% of overall cancer cases[3].	54% of melanoma
	11% of colorectal cancer

Mutant peptides are not centrally tolerized;

cognate TCRs are present in naïve repertoire[13-16]

Mutant p53- and BRAF-specific T cells are

known to mediate anti-tumor efficacy[14,15]

		T Cell IFNγ Response: Spleen Day 21		T Cell IFNγ Response: Spleen Day 21
splenocytes		Stim: BRAF V600E OLPs	splenocytes	Stim: BRAF V600K OLPs
1200				1000
900				800
6x	5x	11x	4x	7x	6x
				600
6					6
1x10	600				1x10
				400
/					/
SFC	300				SFC

Antigen	Mock	18mer	30mer	Antigen	Mock	18mer	30mer
10mer	10mer

Longer antigen peptides contain greater numbers of potential epitopes, increasing the probability of a strong immune response in various HLA haplotypes.

Potent T cell responses are observed after only two doses with AMP-p53 R248W, which are further improved upon after a third bi-weekly dose.

					200
IFNγ					IFNγ
0					0
Peptide		SOL AMP	SOL AMP SOL AMP	Peptide		SOL AMP	SOL AMP SOL AMP
Mock			V600E +	Mock			V600E +
Antigen	V600E	V600K	Antigen	V600E	V600K
	V600K		V600K

T cell responses are detectable after only two doses of the AMP-BRAF V600E, V600K, and combination (ELI-007) vaccines.

	T Cell IFNγ Response: Spleen Day 35		T Cell IFNγ Response: Spleen Day 35
splenocytes	Stim: BRAF V600E OLPs	splenocytes	Stim: BRAF V600K OLPs
20000			15000
15000
			10000
10000	24x	18x	10000	27x	10x
16x	11x
6	7500			6	7500
1x10			1x10
5000			5000
/			/

		12000
6		10000
/SFCCytokine1x10	splenocytes
8000
		6000
		4000
		2000
		0

Peptide

Antigen

Overlay

Percent Polyfunctional

T Cell Cytokine Response: Spleen Day 35

Stim: p53 R248W OLPs

Triple+

IFNγ+ IL2+

IFNγ+ TNFα+

IL2+

TNFα+

IFNγ+

Mock	SOL	AMP	SOL	AMP	SOL	AMP
10mer	18mer	30mer

1x10/SFCCytokine	lymphocytesresident-lung	8000
6
		6000
		4000
		2000
		0
	Peptide
	Antigen

Overlay

Percent Polyfunctional

T Cell Cytokine Response: Lung Day 35

Stim: p53 R248W OLPs

Triple+

IFNγ+ IL2+

IFNγ+ TNFα+

IL2+

TNFα+

IFNγ+

Mock	SOL AMP SOL AMP SOL AMP
	10mer	18mer	30mer

1 Cytokine

2 Cytokines

3 Cytokines

SFC	2500					SFC	2500
IFNγ	0					IFNγ	0
Peptide		SOL AMP	SOL AMP SOL AMP	Peptide		SOL AMP	SOL AMP SOL AMP
Mock			V600E +	Mock			V600E +
Antigen	V600E	V600K	Antigen	V600E	V600K

Vaccine:

5000

pg/ml

		Multiplexed Immune Proteomics: Spleen Day 35
			Stim: p53 R248W OLPs
R248W 10mer		R248W 18mer			R248W 30mer
		200	30000			2000	100000
Mock	SOL	AMP	Mock	SOL	AMP		Mock	SOL	AMP
							80000

2500

2000

[IL2,

The AMP-Platform Delivers Cargo Directly to the Lymph Nodes[17,18]

Smart trafficking to the lymph nodes after subcutaneous dosing generates immune responses with increased magnitude, function, and durability.

Takes advantage of potent lymph node immune mechanisms, including activation of innate and adaptive immune cells, antigen-spreading,and improved tumor T cell trafficking / infiltration.

Mutant p53/BRAF peptides provide validated antigens for application of the Amphiphile platform.

Lymph node delivery of potent adjuvants minimizes systemic exposure to improve safety.

Lymph Node Biodistribution

AMP Platform Design

120

O

O

x

Nodes

Antigen

Albumin Binding Lipid

Peptide Antigen

PEG Linker

100

Albumin

O

(65 kDa)

O

Lymph

80

60

O

Dose% in

40

Adjuvant

NH

O

NH

20

Peptide Antigens

Albumin Binding Lipid

CpG-DNA

0

Molecular Adjuvants

0

20

40

60

80

Molecular Weight (kDa)

AMP MOA

Tissue Injection Site

Lymph Node

Amphiphiles

Endogenous Albumin

Albumin-bound Amphiphiles

1 Subcutaneous	2	Albumin	3	Lymph node	4	Adaptive
injection		binding		targeting		immune response

Biodistribution of AMP Vaccines[17]

			Soluble CpG		AMP-CpG
2			T Cells B Cells		T Cells B Cells

			0h	24h
1					lymph node	lymph node
			3.3nmol
			AMP-CpG-FITC or
			Soluble CpG-FITC

Clinical Experience[19,20]

ELI-002,an AMP-vaccinetargeting mKRAS, is currently being investigated in clinical trials for treatment of pancreatic and colorectal cancer (AMPLIFY-201: NCT04853017 and AMPLIFY-7P: NCT05726864).

ELI-002has shown preliminary safety / tolerability, and significant increases in mKRAS T cell responses, associated with reduction / clearance of tumor biomarkers, and reduced risk of progression and death.

Vaccine candidates targeting mutant p53 / BRAF have been built from the AMP-platform, and utilize the ELI-002 adjuvant, AMP-CpG, providing the potential for favorable clinical tolerability and activity.

V600K

V600K

Further immunization with the AMP-BRAF vaccines increases the T cell response substantially.

Polyfunctional T Cell Responses: Cytokines and Granzyme

Left Y-Axes		4000
γ, GzmB]	3000
2000
	[IFN	1000
		0

				150						1500
					20000
											60000
				100						1000
											40000
					10000
				50						500	20000
			GM-CSF	0	0			GM-CSF		0	0			GM-CSF
IFNγ	TNFα	IL2	GzmB	IFNγ	TNFα	IL2	GzmB		IFNγ	TNFα	IL2	GzmB
Mock				2	Mock					2	Mock				2
			1					1				1
SOL				0	SOL					0	SOL				0
				-1						-1					-1

1500

1000

500

0

Baseline Average Z-

TNFα, GM-CSF]	Right Y-Axes
pg/ml

Rel. Protein

Multiplexed Immune Proteomics: Spleen Day 35

Stim: BRAF V600 OLPs

AMP																											AMP																								AMP
									-2																-2									-2

Subtracted score

Expression

Vaccine:

	80000	Mock
Axes-YLeftpg/mlGzmB],γ	60000
15000
	10000
[IFN	5000
	0
		IFNγ

Mock

SOL

AMP

V600E

SOL

	TNFα		IL2			GM-CSF

			V600K				V600E + V600K
1500							1500
AMP	40000	Mock		SOL		AMP		40000	Mock		SOL		AMP
	10000							10000
1000							1000
	7500
500	5000						500	5000
	2500
0	0				GM-CSF		0	0				GM-CSF
GzmB		IFNγ	TNFα	IL2	GzmB			IFNγ	TNFα	IL2	GzmB
1 . 5		Mock				1.5			Mock				1.5
0		SOL				0			SOL				0
- 1 . 5		AMP				-1.5			AMP				-1.5

400

300

200

100

0

Average ZBaseline Subtracted -score

[IL2, TNFα, GM-CSF]	Right Y-Axes
pg/ml

Rel. Protein Expression

Polyfunctional T cells reside not only in secondary lymphoid tissues like spleen, but in peripheral organs such as the lung, which is one of the predominant sites for metastatic spread in melanoma and colorectal carcinoma.

AMP-p53 R248W Generates Cytotoxic T Cells

		T Cell Cytotoxic Response: Spleen Day 35			In Vivo Target Cell Killing: Spleen Day 35
splenocytes		Stim: p53 R248W OLPs				Stim: p53 R248W OLPs
20000					80	54%	46%
				Killing				Low [Tag-it Violet]
							Unspecific Targets
15000	36x	14x	20x	60			High [Tag-it Violet]
				p53-specific Targets
6					% Target Cell
SFC / 1x10	10000				40	Mock
5000				20	52%	48%	74%
									26%

		Cytotoxic Response: Spleen Day 35			Cytotoxic Response: Spleen Day 35
		Stim: BRAF V600E OLPs				Stim: BRAF V600K OLPs
cells	12				cells	8
CD3+		2.4x	2.7x	1.9x	CD3+	6		2.1x	2.2x	2.4x
among	8				among
				4
GzmB+%	4				GzmB+%
				2
	BL					BL
	0					0
Peptide	SOL AMP SOL AMP SOL AMP	Peptide		SOL AMP	SOL AMP	SOL AMP
Mock		V600E +	Mock			V600E +
Antigen	V600K	Antigen	V600E	V600K
V600E	V600K		V600K

Polyfunctional T cells induced by AMP-BRAF vaccines produce high levels of cytokines as well as cytolytic GzmB.

➢ AMP enhances vaccine potency via targeted lymph node delivery.

MESSAGES	➢ ELI-007 and ELI-008 substantially improved T cell responses over
		soluble comparator vaccines:
		• Polyfunctional T cells that produce TH1-associated cytokines:
HOME		(IFNγ / TNFα / IL2 / GM-CSF).
	• Secretion of Granzyme B, potent cytolytic function
➢ AMP-vaccines have the potential to address a high, unmet medical
TAKE		need for millions of patients with BRAF / p53 mutations annually.
➢	The AMP-platform technology is simple, rapid and scalable for broad

clinical application.

GzmB	0				0
Peptide	SOL AMP SOL AMP SOL AMP	SOL AMP
Antigen	Mock	18mer	30mer	Mock	SOL	AMP
10mer	18mer

AMP-p53 R248W generates GzmB producing, cytotoxic T cells capable of killing p53 R248W-pulsed target cells in vivo.

AMP-vaccination Generates Immunity to Common p53 Mutants

T Cell IFNγ Response: Spleen Day 35 Stim: respective p53 OLPs

	R248W			R175H			R273H			G245S
splenocytes	25000			splenocytes	8000			splenocytes			splenocytes	2000
								6000
					6000
	15000	10x			3000	70x			3000	100x		1500	7x
6				6				6			6
1x10/	10000			1x10/	2000			1x10/	2000		1x10/	1000
SFCγ	5000			SFCγ	1000			SFCγ	1000		SFCγ	500
IFN	0			IFN	0			IFN	0		IFN	0
	Mock	SOL	AMP			Mock SOL	AMP		Mock	SOL AMP			Mock SOL	AMP
splenocytes		Y220C	splenocytes		C135Y	splenocytes	R158H	splenocytes		H214R
								15000
	21000				25000
		18x				5x			2000	37x			8x
	10000			20000
										10000
6	7500			6	15000			6			6
1x10			1x10			1x10			1x10
5000			10000			1000
/			/			/			/	5000
SFC				SFC				SFC			SFC
2500			5000
IFNγ			IFNγ			IFNγ			IFNγ
0			0			0		0
	Mock SOL	AMP			Mock SOL	AMP		Mock SOL AMP			Mock SOL	AMP

8 candidate p53 mutant 30mer AMP-peptides elicit strong T cell responses upon immunization, demonstrating the versatility, ease of application and potency of the AMP-platform.

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