Keystone Symposium: B cells and TLS_POSTER

Inhibition of Semaphorin 4D in combination with immune checkpoint therapy induces organized lymphoid structures within the tumor microenvironment that correlate with clinical outcome

#3012

Introduction Myeloid cells play a critical role in suppression of adaptive immunity within the TME. Semaphorin 4D (SEMA4D) signaling through its receptors (PlexinB1/B2, CD72) excludes activated antigen presenting cells and promotes recruitment and suppressive function of myeloid suppressor cells (MDSC)(1). In preclinical and clinical studies, SEMA4D antibody blockade increased penetration of B cells, antigen presenting dendritic cells (DC) and T cells into the tumor and attenuated MDSC in the TME, leading to enhanced efficacy of immune checkpoint inhibitors (ICI) (2).

Data presented here support the HYPOTHESIS that SEMA4D blockade regulates crosstalk of immune cells in TME to promote organized functional immune interactions as a novel mechanism of immune enhancement.

Background

Pepinemab combined with immune checkpoint inhibitors (ICI) appeared to induce mature TLS that correspond with recurrence free survival. In contrast, patients treated with nivolumab alone demonstrated few B cells and disorganized T cells (4) following neoadjuvant treatment in patients with Stage III metastatic melanoma (NCT03690986).

RESULTS: R/M HNSCC				Immune Aggregates correlate with PFS
KEYNOTE-B84 interim analysis	SEMA4D	Anti-SEMA4D		Longer progression free survival (PFS)
	with presence of B cell aggregates
				Mechanisms of	Mechanisms of
				survival
Spatial analysis of immune cells in TME	Immune Suppression	Immune Enhancement	100
Tumor expresses SEMA4D
revealed	that	combination	therapy
and PD-L1 to survive	Immune Attack /
induced	highly	organized	immune		free
	Tumor Cell Death

CD20 Ki67 CD4

Pepi

Nivo

Ipi

pCR

Pepi

Nivo

pCR

Pepi

Ipi

mPR

CD8

	# TLS Correlates with
A. Recurrence-free survival
Intratumor	0.8		✱
	✱
0.6
0.4
#TLS/mm^2
0.2
	0.0	recurrence No recurrence
	No
	recurrence	recurrence
	Pepinemab	NO
	pepinemab
	treatment
	treatment
B.	Pepi + Nivo + Ipi
	100
Survival				*
of			Pepi+Ipi
	50

aggregates localized within HNSCC tumor					-Progression	50	B cell Aggregates
					**
PLXN binding to
bed with high density of activated B cells,		T cells
SEMA4D induces
DC's, CD4+ and CD8+ T cells, including	recruitment and		cannot kill
	tumor			No B cell Aggregates
stem-like CD8+TCF1+PD1+ T cells.	suppressive			0
function of
				0	5	10
	myeloid cells 1		Pepinemab
Illustrations created using BioRender					Time(months)
			activates DC to
		Myeloid cells	anti-PD-1/PD-L1	B cell aggregates correlate with PFS. On-treatmentPatient
		organize immune
		inhibit T cell activity	biopsies with one or more B cell aggregates positively
		aggregates and	activates T cell activity
			correlates with	longer progression-free survival. N=12 on-
Dendritic cells within TME			expand T cells		treatment biopsies at interim analysis. Log Rank survival
					statistical analysis resulted in a ** p value of 0.0056.

CD8 T cells within TME

CD11c+ DC	Induction of B cell Aggregates within TME
CD8+ T cells

Nivo

mPR

Pepi+Nivo (pCR)

robabilityP		Nivo		Pepi+Nivo
0
0	5	10	15	20
	Time to Recurrence (Months)

Nivo (mPR)

TME	1000	P = 0.0640
800
/mm^2
600
CD11c+	400
Density	200
0
		PreTx OnTx	PreTx OnTx
MacDC		DC1
			P = 0.0983

	2500	✱
TME	2000
m^2
1500
/m
CD8+	1000
Density	500
	0

PreTx OnTx PreTx OnTx

CD4 T helper cells

B cells within TME

Mature B cell aggregates were induced with pepinemab plus pembrolizumab treatment

Pre-Treatment Biopsy

On-treatment Biopsy

B cells

Disease Control

Progressive Disease

Disease Control

Progressive Disease

● CPS <20

CPS

●○●●○○○○○●●●●●○●●○○●○○○●●○

● ● ● ● ● ● ● ● ● ○ ○ ○

500

HPV - - - - - - - - - - -

+ - - - - - + - - - - - - - -

- - - + - - -

- - - - -

○ CPS ≥20

TME/mm^2 CD20

400

Aggregate

400

B cell

300

300

DC		CD8 T cell		CD4 T helper cell		B cells
All CD11c+		CD8+		CD4+FoxP3-		CD20+

Melanoma patients received 2 doses of immunotherapy (Day1&21) before surgery (day 35-49), n=8/group. Data from resected tumors is shown. Investigator sponsored trial NCT03690986, in collaboration with Emory University A. Pepinemab treatment was associated with higher density TLS and no recurrence. = no treatment. Statistical analysis: Two tailed unpaired t test, P<0.05 B. Pepi combinations result in durable recurrence-freesurvival compared to Nivo alone. Statistical analysis: Log-rank(Mantel-Cox) test P=0.0268. pCR=pathologic complete

TME	500	✱		TME	150
/mm^2	400			^2/mm
			100
Density CD11c+CD68+	300			CD11c+CD141+ensity D
200			50
100
0			0
	PreTx OnTx	PreTx OnTx	PreTx OnTx PreTx OnTx

TME/mm^2	3000
)	2000
-CD4(FoxP3	1000
Density	0
	PreTx OnTx PreTx OnTx

	200																																																																									>Aggregate20
Density	100	200
0	Bcells/
PreTx OnTx PreTx OnTx
		100
		20
		1	2		1		2		3		4		5		1		2		3		4		6		8		9		0		1		1		2		4		5		6		7		8		9		0	1
		0	0		0		0		0		0		0		0		0		0		0		0		0		0		1		1		0		0		0		0		0		0		0		0		1		1
				R		R		R		R		R		D		D		D		D		D		D		D		D		D		D		D		D		D		D		D		D		D		D		D
		CR CR P		P		P		P		P		S		S		S		S		S		S		S		S		S		P		P		P		P		P		P		P		P		P		P

response; mPR=major pathologic response

Disease Control		Disease Progression

N=26

N=12

METHODS	Sponsor: Vaccinex
Pembrolizumab provided by: Merck Sharp & Dohme Corp.
KEYNOTE-B84 R/M HNSCC Clinical Trial Design
➢ Recurrent & Metastatic HNSCC	Phase 1b	Phase 2	Outcome Measures
➢ Immunotherapy naïve
➢ Enroll both PD-L1 high (CPS≥20)		Pre-specified	Safety
& PD-L1 low/neg (CPS<20)	Safety

Increase in CD11c+ dendritic cell populations and CD8 T cells in pathologist-definedtumor area (TME) following treatment is associated with disease control (CR, PR, SD). Presence of mature B cell aggregates is induced with

treatment, associated with durable disease control, and is unexpected in HPV-negativeHNSCC. Mature B cell aggregates contain >20 B cells (5). B cell aggregates appear to be induced by treatment, as only 31% (5/16) of Pre-Tx biopsies among Disease Control tumors contained mature B cell aggregates, while 100% (7/7) contained mature aggregates following treatment. Ϯ designates biopsies from distal metastasis to lung, all other samples are local to head and neck regions. Disease Control (includes CR complete response, PR partial response, SD stable disease) determined by Response Evaluation Criteria (RECIST1.1) Statistical analysis: Two tailed unpaired t test, P<0.05.

Composition of Immune Aggregates

22C3 pharmDx kit (Dako)	Well tolerated	Interim Analysis Objective Response
➢ Treatment: 20mg/kg pepinemab	36 patients	Biomarker Outcomes
+ 200mg pembrolizumab, Q3W

NCT04815720

IHC Biomarker Analysis

Biopsies collected pre-treatment screening and at week 5 cycle 3

B cell aggregates are highly organized with key immune cells for antigen presentation and expansion of T cells

Disease Control

Progressive Disease

Durable disease control is associated with a higher percentage of DC's within TLS following treatment

Disease ControlProgressive Disease

●

●

●

●

●

●

●

●

●

○

○

○

CPS

-

-

-

+

-

-

-

-

-

-

-

+

HPV

TCF1+PD1+CD8+ Stem-like progenitor cells are associated with Disease Control and located within B cell aggregates

Stem-like CD8	Stem-like CD8 T cells are located within
within TME	B cell Aggregates

1. Stain → Image → Strip → Stain ~14 staining cycles per slide

Lymphocyte	APC Panel	Myeloid
Panel		Panel
Hematoxylin	Hematoxylin	Hematoxylin
Sema4D	Sema4D	CD33
PD-1	CD163	CD15
CD69	CD11c	CD14
CD8	HLA-DR	Arg1
CD4	CD68	HLA-DR
FoxP3	CD141	Sema4D
CD26	CD206	S100A9
CD20	Arg1	CD16
CD39	PanCK	PanCK
CD45	PD-L1	PD-L1
TCF1

4. Image analysis software, Visiopharmalgorithms were written and automated to identify cell phenotypes, quantify density within entire tumor area, neighborhoods.

5. Unbiased Software algorithm identifies B cell

aggregates using heatmaps. Heatmaps look at B cells clustering within 50um of each other.

6. B cell aggregates classified by:

Low Density B cells

(<20 cells)

High Density (Mature) B cell aggregate(5,6)

(≥20 cells)

DC		CD8 T cell		Stem-Like T cells		CD4 T helper cell		Treg		B cells
All CD11c+		CD8+		CD8+ TCF1+		CD4+FoxP3-		CD4+FoxP3+		CD20+

● CPS <20 ○ CPS ≥20

TMED1+CD8+cells/mm^2 TCF1+P	5	✱✱✱	aggregatecell B/ cellsCD8+TCF1+PD1+	20						Disease Control							Progressive
														Disease
4
		15
3
2		10
1		5
0		0
	Disease	Disease			3			2		3		4		6		9		1		2			4		5			6		9
		0			0		0	0	0	0	1	0			0		0		0	0
				R			R			D		D		D		D		D		D			D			D		D		D
	Control	Progression		C		P			S	S	S	S	S	P		P			P	P		P

PanCK

Ki67

1. Virtually align stains by panel using VisiopharmTissuealign
2. ROI drawn around pathologist identified tumor area, includes tumor and tumor-associatedstroma

1. Expand B cell hubs by 150um to identify cells interacting with B cells.
2. Classification of cells within Immunity Aggregates

Patients experiencing disease control following treatment with pepinemab plus pembrolizumab showed an increase in the number of B cell aggregates	Stem-like CD8 are associated with improved response to immunotherapy
(above). These aggregates exhibit spatial organization that is characteristic of functional immune response, similar to mature TLS. Highly organized immune	and share features with TFH cells found within immune aggregates.
aggregates contain zones of high density APCs (activated DC, B cells) and a T cell zone with CD8, CD4 T helper cells and stem-like CD8's. In contrast, patients	Patients with disease control had a higher density of stem-like CD8 T cells
with progressive disease and untreated patient tumors predominantly contain no or few immune aggregates with spatial interactions that favor immune	after treatment within the TME. Stem-like CD8 T cells are located within B
suppression, including abundance of Treg.	cell aggregates. Statistical analysis: Two tailed unpaired t test, P<0.05

CONCLUSION

Pepinemab plus pembrolizumab		Immune Aggregates
induce B cell aggregates in		correlate with
patients experiencing disease		Progression Free
control	survival		Survival (PFS)
	100
	Progression-free		B cell
	50	Aggregates
	N=7	**
			No B cell Aggregates
		0	N=5
		0	5	10
Disease	Disease		Time(months)
Control	Progression
N=7	N=4

B cell aggregates are induced in hard-to-treat HPV neg patients with disease control

10/11 matched pre to on treatment biopsies were HPV-

HPV+

HPV-

Pepinemab plus Pembrolizumab showed ~2x increase in ORR and PFS in hard-to-treatPD-L1 low tumors compared to historical response rate for checkpoint monotherapy in this population

		PD-L1 Low
		(CPS < 20)
	KEYNOTE-B84	KEYNOTE-048 (7)
	pepi + pembro	pembro
Total		(19)	(168)
CR	2	10.50%	2.40%
PR	2	10.50%	9.50%
SD	10	52.60%	25.00%
ORR*	4	21.10%	11.90%
DCR	14	73.70%	36.90%
PFS, months		5.79	2.2
(95% CI)		(2.2 - NR)	(2.1 - 2.9)

Results suggest that pepinemab combined with ICI induced formation of highly organized lymphoid aggregates in the tumor with a high density of activated B cells, DC and T cells including TCF1+PD1+CD8+ stem-like progenitors. Together with similar observations indicating that combination immunotherapy with pepinemab induces mature lymphoid structures in tumors of patients with metastatic melanoma, provides evidence of treatment-induced biologic activity corresponding with disease control and suggests a novel and independent mechanism of pepinemab to enhance immune interactions and activity of ICI in resistant settings.

REFERENCES

1. Clavijo PE et al. Cancer Immunol Res. 2019 (2):282-291.
2. Shafique MR et al. Clin Cancer Res. 2021 Jul 1;27(13):3630-3640.
3. Gong et al. Molecular Cancer (2023) 22:68.
4. Olson B et al. Journal for ImmunoTherapy of Cancer 2022;10.
5. Ruffin AT et al. NATURE COMMUNICATIONS (2021) 12:3349.
6. Labroots webinar: Tertiary lymphoid structures to the forefront of immunotherapy: what are they good for? Tullia C. Bruno, PhD Assistant Professor,
   University of Pittsburgh, Hillman Cancer Center
7. NCT02358031. Burtness et al. 2022 Clinical Oncology 40 (21): 2321-2332.

NOTE: CPS <20 was calculated post-hoc from analysis of CPS<1 and 1-19 assessments; these do not represent alpha controlled analyses.

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