Caribou Biosciences : CB-012 IND-enabling preclinical data AACR 2024

(TKO: triple knockout,

Abstract 6323

Preclinical evaluation of CB-012, an allogeneic anti-CLL-1CAR-T cell therapy, that exhibits specific and

potent cytotoxicity in acute myeloid leukemia (AML) xenograft models

Brian Francica1, Elizabeth Garner1, Sai Namburi1, Cian Colgan1, Tristan Fowler1, Devin Mutha1, Art Aviles1, Morena Stanaway1, Raymond Guo1, Zili An1, Erin Kelly1, Émilie Degagné1, George Kwong1, Leslie Edwards1,

Emma Jakes1, McKay Shaw1, Benjamin Schilling1, Jeremy Huynh1, Ricky Luu1, Max Sidorov1, Rhonda Mousali1, Mikk Otsmaa1, Peter Lauer1, Justin Skoble1, Steven Kanner1

1Caribou Biosciences, Inc., Berkeley, CA

ABSTRACT

CB-012 demonstrates antigen-specific cytotoxicity, proliferation, and cytokine secretion against CLL-1-expressing AML cell lines

Unbiased screen for off-target binding reveals high specificity of CB-012 scFv

Background

CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023.

Methods

Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in

A

B

Target	Effector
HL-60	CB-012
HL-6CLL-1 KO	CB-012
HL-60	TKO

E:T Ratio

Target	Effector
THP-1	CB-012
THP-1CLL-1 KO	CB-012
THP-1	TKO

C

D

HL-60

HL-60CLL-1 KO

THP-1

THP-1CLL-1 KO

T Cells Only

		Cell Trace Violet
HL-60	HL-60CLL-1 KO	THP-1	THP-1CLL-1 KO	T Cells Only

CB-012	TKO

Figure 2: A,B) 24-hourin vitro cytotoxicity assay utilizing target cell lines HL-60 or THP-1 and their respective isogenic CLL-1 KO cell lines. C) Cell proliferation assay utilizing cell trace violet staining to track cell divisions 72 hours after stimulation of CB-012 or TKO with labelled target cells or with no target cells ("T cells only"). D) Cytokine production by CB-012 or TKO T cells after 24 hours of in vitro stimulation with target cells.

T cells engineered with a TCR KO, B2M KO, and PDCD1 KO)

A

6500+ cDNAs spotted	Reverse transfection of overlaid	CB-012 scFv applied to arrays
on specialized slides	human cells results in over-	and binding detected
	expression of individual proteins

		Specific protein target(s) identified	Hit
				validation
				& specificity
B				testing
CB-012anti-CLL-1 scFv	Control anti-CTLA-4 scFv	using
	negative

controls

C	Specific Interactions
Test
Found
PBS (-control)	No
CTLA4 (+control)	Yes
CLEC12a (CLL-1, +Experimental control)	Yes
6,105 human plasma membrane proteins	No
400 heterodimers	No

Figure 5: A) Schematic of workflow for the Retrogenix unbiased screen for off-target scFv interactions. A library screen was performed testing the CB-012anti-CLL-1 scFv interactions across a cell array over-expressing 6,105 individual full-length human plasma membrane proteins and cell surface-tethered human secreted proteins as well as a further 400 human heterodimers. B) Spot array results from the confirmation screen. All identified library interactions were re-expressed and probed with CB-012anti-CLL-1 scFv to determine which interactions may be specific to the CB-012 scFv. All positive results were also positive in the anti-CTLA-4

vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential.

Results

CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that

(pg/mL)	600
400

	300		2000		400		800
(pg/ml)		(pg/ml)	1500	(pg/ml)	300	(pg/ml)	600	Target only
200	TKO

CLEC12A = CLL-1

CD86 = CTLA-4

control group except for CLL-1.C) Summary of results from screen.

E:T Ratio

IL-2	200
	0

	1000	A	200	B	400	CB-012
	Granzyme
100
TNF-	500	100	Granzyme	200
IFN-
0	0		0		0

CB-012 pharmacokinetics in tumor naïve murine models demonstrates limited antigen-independent expansion

THP-1THP-1	THP-1	THP-1	THP-1THP-1	THP-1THP-1	THP-1THP-1
CLL-1 KO		CLL-1 KO	CLL-1 KO	CLL-1 KO	CLL-1 KO

Human cells/μg mouse genomic DNA

6000

4000

2000

600

400

200

40

30

Bone Marrow

6000

4000

2000

600

400

200

40

30

Liver

6000

4000

2000

30

20

Heart

6000

4000

2000

30

20

Skeletal Muscle

the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non- specific interactions. CB-012CAR-T cells exhibited limited tissue infiltration and expansion in treatment

PDCD1 KO in CB-012 confers significant increase in activity

20

10

0

6000

D3 D8 D22 D36 D50 D85

Scheduled Euthanasia Day

20

10

0

6000

D3 D8 D22 D36 D50 D85 Scheduled Euthanasia Day

10

0

6000

D3 D8 D22 D36 D50 D85 Scheduled Euthanasia Day

10

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6000

D3 D8 D22 D36 D50 D85 Scheduled Euthanasia Day

naïve, immunocompromised murine models.

Conclusion

CB-012, the first allogeneic anti-CLL-1CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo.

A	B
			1×1010	Vehicle
25	BioluminescentIntensity	(Photons/s)	1×109	BioluminescentIntensity	(Photons/s)
20	1×108
15	1×107
PD1+-	1×106

1×1010		PDCD1 WT

1×109

1×108

1×107

1×106

				C
BioluminescentIntensity		1×1010	CB-012	CB-012
(Photons/s)	1×109
1×108
1×107
1×106

Human cells/μg mouse genomic DNA

4000

2000

600

400

200

40

30

20

10

0

Lung

D3 D8 D22 D36 D50 D85

Scheduled Euthanasia Day

4000

2000

600

400

200

40

30

20

10

0

Kidney

D3 D8 D22 D36 D50 D85 Scheduled Euthanasia Day

4000

2000

600

400

200

40

30

20

10

0

Blood

D3 D8 D22 D36 D50 D85

Scheduled Euthanasia Day

4000

2000

600

400

200

40

30

20

10

0

Spleen

D3 D8 D22 D36 D50 D85

Scheduled Euthanasia Day

Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044).

CB-012 is an anti-CLL-1 allogeneic CAR-T cell therapy with a PD-1 knockout and immune cloaking

4 Armored with 5 genome edits

		B2M
		KO		TRAC gene knockout (KO)
			1
			• Eliminates TCR expression, reduces GvHD risk
		PD-1 KO		Human anti-CLL-1 CAR site-specifically inserted into TRAC gene
	Anti-	3	2
	CLL-1	• Eliminates random integration, targets tumor antigen
	B2M-HLA-E
	CAR
	2	5		PD-1 KO for enhanced antitumor activity
TCR	PD-L1	3
	• Potentially better therapeutic index via initial tumor debulking
KO	CLL-1

1	B2M gene KO
	4
	• Reduces HLA class I presentation and T cell-mediated rejection
	B2M-HLA-E-peptide fusion site-specifically inserted into B2M gene
	5
	• Blunts NK cell-mediated rejection

1st CAR-Twith checkpoint inhibition	Cas12a chRDNA editing for	Potent, fully human anti-
and immune cloaking
reduced off-target editing and	CLL-1 scFv 2 with a CD28
(PD-1 KO, B2M KO + B2M-HLA-E-peptide
enhanced insertion rates	costimulatory domain
fusion) to enter the clinic1

1. To Caribou's knowledge
2. Anti-CLL-1-specificscFv exclusively licensed from Memorial Sloan Kettering Cancer Center for allogeneic cell therapies

ABBREVIATIONS:
AML: acute myeloid leukemia	RP2D: recommended phase 2 dose
MTD: maximum tolerated dose	SCT: stem cell transplant

RDE: recommended dose or doses for expansion TKO: triple knockout, T cells engineered with a TCR KO, B2M KO, and PDCD1 KO

CD8

10																			1×105
%
																		0	10	20	30	40
5
																							Days Post Treatment
0
				T cell only	PMA	10					Vehicle
													Ionomycin
					Target Condition

1×105

0

10

20

30

40

Days Post Treatment

20PDCD1 WT

1×105
0	10	20		30	40
						Days Post Treatment
																PDCD1 WT
20						CB-012

Figure 6: Non-tumorbearing NSG mice were treated with 30 x 106 CB-012T cells. Persistence of cells in labelled organs was measured by sacrificing animals, processing organs, and detecting CB-012-specificsequences by ddPCR over an 85-daytime course.

	Change	5
PDCD1 WT	0
	%
CB-012
BW	-5

-10

0

10

20

30

40

Change	10
0
BW %
-10

-20

	0	10		20	30	40

Change	10
0
BW %
-10				Counts
	-20				PD-1
	0	10	20	30	40

CB-012 AMpLify Phase 1 trial design

r/r AML

Lymphodepletion CB-012

Figure 7:

Patients with r/r AML

Days Post Treatment

Days Post Treatment

Days Post Treatment

Figure 3: A) PD-1expression was analyzed by flow cytometry on fully edited CB-012 ("CB-012")or CB-012that was not edited at the PDCD1 locus ("PDCD1 WT") after 24 hours in culture +/- PMA/ionomycin stimulation. B) In vivo tumor burden as measured by luminescent intensity (top) and corresponding mouse body weight (BW%, bottom). 5 x 105 HL-60cells edited to express GFP and luciferase as well as to overexpress PD-L1were engrafted by intravenous injection and allowed to establish for 7 days before injection of 1 x 107 CB-012or PDCD1 WT CAR-Tcells. C) Ex vivo expression of PD-1on CB-012or PDCD1 WT T cells extracted from a terminal take down of bone marrow from surviving mice 42 days after treatment with each cell type. Data represents the concatenation of transferred edited T cells from 5 mice.

-5 to -3DAYS	DAY 0	28 DAYS	3 MONTHS	6 MONTHS	9 MONTHS	12 MONTHS
				Safety and tolerability
Cyclophosphamide				Response assessment
(750 mg/m2/d)	SINGLE
Fludarabine	DOSE	Dose level 1: 25 x106	CAR-T cells (enrolling patients)
(30 mg/m2/d)
Part A: 3+3 dose escalation - enrolling	Part B: dose expansion
• Objective: safety, determine MTD/RDE	• Objective: antitumor response, determine RP2D, safety

•	Relapsed or refractory AML
	patients should have received
	at least 1 but not more 3 prior
	lines of therapy
•	Patients with prior allo or auto
	SCT are allowed
•	Exclusions: prior CAR-T cell
	therapy and/or CLL-1-targeted
	therapy

CB-012 displays minimal off-target,off-tissue activity

CONCLUSIONS

TKO control T cells

CB-012CAR-T cells

• CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and

5:1 Effector Cells:Targets

10:1 Effector Cells:Targets

20:1 Effector Cells:Targets

1KO

Cells

-VSMC

-

Lu

-60

-60

CLL

3

-5i

1

-

18

B

Tes

OVCAR3HA

THLE

CCD

HBEC Primary

-293 .

NIH

HL

HL

90196BHEK

Hs

T/G

100

50

0

-50

1KO

Cells

-

3

Lu

-5i

CLL

18

B

-293

-60

-60

THLE

CCD

HL

HL

HBEC Primary 90196BHEK

-

	1						-VSMC
	Tes	OVCAR3HA
	.		NIH			T/G
Hs

100

50

0

-50

	leukemic stem cells but is not expressed on hematopoietic stem cells
•	CB-012 is the first allogeneic anti-CLL-1CAR-T cell therapy using checkpoint disruption and immune
	cloaking armoring strategies, engineered with a next generation Cas12a chRDNA technology
•	Specificity of the anti-CLL-1 fully human scFv was demonstrated in an unbiased protein-binding study
	and no adverse safety signals were observed in murine toxicology models
•	A single dose of CB-012 resulted in robust tumor control, leading to significant prolongation of survival
	in AML xenograft models
•	Data from these preclinical studies supported the IND clearance of CB-012 by the FDA in October 2023

Figure 4: 9 cell lines representative of vital human tissues as well as positive and negative control cell lines (HL-60and HL-60 CLL-1KO, respectively) were incubated in vitro for 48 hours with 3 effector-to-targetratios to define potential off-tumoractivity of CB-012.Cytotoxicity profiles or experimental cell lines were similar when cell lines were incubated with CB-012as compared to cultures incubated with TKO. (TKO: triple knockout, T cells engineered with a TCR KO, B2M KO, and PDCD1 KO)

CORRESPONDING AUTHOR:

• A Phase 1 first-in-human clinical trial (AMpLify) in relapsed/refractory AML patients is ongoing and

currently enrolling patients in the United States (NCT06128044).

Brian Francica, PhD, Caribou Biosciences, Inc. ([e-mail])	AACR ANNUAL MEETING
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