Introduction

We have established over 200 CDX models with validated tumor growth curves, covering major cancer types such as colon, lung, breast, and pancreatic cancers. Contact us for more detailed data.

Results

Case Study 1: Successfully establishing a CDX lymphoma model and verifying the efficacy of antibody drugs with B-NDG mice

A Raji lymphoma mouse model was established using B-NDG mice and the efficacy of antibody X was verified. B-luc-GFP Raji cells (5×10⁵) were injected via tail vein of B-NDG mice and divided into 3 groups: no dosing group, day 10 dosing group, day 3 and day 10 dosing group. Both dosing groups were given the same dose of antibody X and tumor growth was observed with imaging at different time points. (A) Imaging of mice to observe tumor growth; (B) Fluorescence intensity curve of tumor cells. The results showed that early antibody therapy (started 3 days after Raji cell transplantation and re-administered 10 days later) was significantly more effective than late antibody therapy (started 10 days after Raji cell transplantation). B-NDG mice is a powerful model for efficacy verification of anti-human antibodies. Values are expressed as mean ± SEM.

Case Study 2: Successfully establishing CDX model and verifying the efficacy of ADC drugs with B-NDG mice

Comparing three cell lines with different Her2 expression level and establishing CDX models in B-NDG mice to verify the difference in efficacy of Her2-targeting ADC drugs. A431, BT-474 and NCI-H1975 cells were implanted subcutaneously in B-NDG mice. Mice were grouped when tumor volume reached to a suitable size, at which time they were treated intravenously with ADC drugs. The drug synthesized internally were marked with “in house”, while the commercial drugs were marked nothing after the drug name. (A) Schematic diagram of CDX model establishment and drug delivery strategy; (B) Expression level of human Her2 in in vitro cultured A431, BT-474, NCI-H1975 cell line; (C) CDX models were established for each of the three cell lines and the efficacy of the Her2-targeting ADC drugs were verified. The results showed that the expression level of human Her2 was high in BT-474 and NCI-H1975 cell line, while the expression level of Her2 in A431 cells was very low. Her2-targeting ADC drugs can significantly inhibit the tumor growth in CDX models established with high-Her2-expressing cell lines (BT-474, NCI-H1975), but had a weak inhibitory effect on low-Her2-expressing tumor (A431). B-NDG mice is a powerful model for efficacy verification of human ADC drugs. Values are expressed as averages ±SEM.

Introduction

Immune checkpoint inhibitors have been game changers in tumor treatment during recent years. With improved therapeutic outcomes observed in a variety of solid tumors, this class of drugs has garnered considerable attention. Lacking of mature T, B, and NK cells, Biocytogen B-NDG mice are worldwide recognized as highly immunodeficient tool mice that are ideal for human cell or tissue transplantation. However, the defective immune system of the model itself makes it impossible to evaluate the efficacy of immunotherapy. Immunodeficient mice reconstituted with human immune cells offer a viable solution to this problem: human immune cells and hematopoietic stem cells are transplanted into B-NDG mice or B-NDG-derived mice to reconstitute their immune system, which can better simulate the human immune system for immunological studies and immune drug evaluation.

Biocytogen currently provides three immune reconstitution models: human peripheral blood mononuclear cell (PBMC) reconstitution, human hematopoietic stem cell (CD34+ HSC) reconstitution, and NK cell reconstitution. The immune reconstitution mouse model aids in the efficacy evaluation of immunotherapy drugs, bolstering the efforts in novel drug development and preclinical evaluation.

Results

Human immune system for PBMC / CD34+ HSC reconstruction

Construction and Application of PBMC Immune Reconstitution Mouse Model

The PBMC-humanized B-NDG mouse model and its variants are a valuable tool for evaluating T-cell-focused studies. We have constructed the following models:

• huPBMC-B-NDG mice model
• huPBMC-B-NDG B2m KO mice plus model
• huPBMC-B-NDG MHC I/II DKO mice plus model
• GvHD-B-NDG mice model

These models support a wide range of studies in immunology and drug development.

Human Immune System Reconstituted Models via Human PBMCs Engraftmen

Engraftment of human PBMCs in B-NDG mice enhances reconstitution of human T cells. Human PBMCs (5E6) were intravenous engrafted into B-NDG mice (female, 6-week-old, n=6). Body weight of each mouse was recorded every week. Death data were collected every day. The peripheral blood was taken at different time points to analyze the reconstitution levels of human immune cells. B-NDG mice showed a high percentage of human CD45+ cells and T cells. B-NDG mice exhibit shortened survival and reduced body weight likely due to GvHD caused by a high Percentages of human T cell reconstitution. Values are expressed as mean ± SEM.

NOTE: There are differences in the level of reconstitution of immune cells due to PBMCs collected from individuals.

When constructing PBMC-humanized models, several critical factors must be considered to ensure successful reconstitution, including donor screening, cell line selection, and inoculation time selection.

1.Donor Screening

PBMCs from various donors had significant difference in body weight, GVHD and survival rate of B-NDG mice

2. Cell Line Selection

PBMC reconstituted mouse CDX model in B-NDG mice

List of CDX huPBMC models

A summary of cancer cell lines that have completed tumorigenic experiments.

Cancer		Cell line
Breast cancer	Breast cancer	MDA-MB-231
Colorectal	Colorectal carcinoma	RKO、HT-29、HCT-8、LOVO、HCT116
Epidermoid carcinoma	Epidermoid carcinoma	A431
Glioblastoma	Glioblastoma	U87-MG
Gastric carcinoma	Gastric carcinoma	NUGC-4、NCI-N87
Hepatocellular carcinoma	Hepatocellular carcinoma	Hep G2
Lung Carcinoma	Lung adenocarcinoma	HCC827、A549、NCI-H292, SHP-77
Lymphoma	Lymphoma	Raji-Luc、B-CAG-hPD-L1 Raji-Luc
		SU-DHL-1、Daudi
Myeloma	Myeloma	NCI-H929
Leukemia	Acute promyelocytic leukemia	HL-60-Luc
	Chronic Myeloid Leukemia	K562
	Leukemia	MV-4-11
Melanoma	Melanoma	A375，SK-MEL-5
Pancreatic carcinoma	Pancreatic carcinoma	MIA PaCa-2

3. Inoculation Time Selection

Treatment of B-NDG mice with different PBMC implantation time point in RKO tumor model using PD-L1 antibody

Treatment of B-NDG mice with different PBMC implantation time point in RKO tumor model using PD-L1 antibody.
RKO cells (5E6), s.c.; Human PBMC cells, i.v.
B-NDG mice (female, 6 week-old, n=6);
Test article: Atezolizumab (10 mg/kg, i.p., BIW×6)
The results showed that the time point of PBMC cells implantation resulted in different antitumor activity.

In vivo efficacy evaluation of a bispecific antibody in B-NDG mice reconstituted with human PBMCs

• NUGC4 cells (5E6), s.c.; human PBMC cells, i.v.
• B-NDG mice (female, 7 week-old, n=6)
• AMG 910 analog: Anti human CD3×Claudin18.2 bispecific antibody

• NCI-N87 cells (1×10⁷), s.c.; human PBMC cells, i.v.
• B-NDG mice (female, 6 week-old, n=6)

• B-luc-GFP Raji cells (5×10⁵), i.v.; human PBMC cells (1×10⁶), i.v.
• B-NDG mice (female, 5 week-old, n=5).

• SHP77 cells (5×10⁵), s.c.; human PBMC cells, i.t.
• B-NDG mice (female, 6 week-old, n=6)

Summary-PBMC Immune Reconstitution Mice Model

Cell line	Cancer type	Mice	CDX based on huPBMC model	Target	Efficacy (Yes/NO，TGI)	Matrigel (Yes/No)
MDA-MB-231	Breast cancer	B-NDG	Succeed	-	-	Yes
RKO	Colorectal cancer	B-NDG	Succeed	PD-L1	43.7%	NO
RKO	Colorectal cancer	B-NDG B2m KO mice plus	Succeed	PD-1×PD-L1	46.6%	NO
HCT116	Colorectal cancer	B-NDG B2m KO mice plus	Succeed	Client project, Bispecific Antibody	NO	NO
Modified HCT116	Colorectal cancer	B-NDG	Succeed	Client project, CD3-TAA	Yes	NO
HCT-8	Colorectal carcinoma	B-NDG	Succeed	CTLA-4	＜5%	NO
A549	Lung cancer	B-NDG	Method Optimization	-	-	-
HCC827	Lung cancer	B-NDG	Succeed	PD-1 (Keytruda)	NO	NO
A375	Melanoma	B-NDG	Succeed	PD-1 (Keytruda)	NO	NO
A431	Epidermoid carcer	B-NDG	Succeed	PD-1 (Keytruda)	NO	NO
B-hPD-L1 Raji-luc	Lymphoma	B-NDG B2m KO mice plus	Succeed	PD-1×PD-L1	Yes	NO
MIA-Paca-2	Pancreatic cancer	B-NDG	Succeed	Client project, Combined therapy	Yes	NO
NUGC-4	Gastric cancer	B-NDG	Succeed	Client project, CD3-TAA	Yes	NO
NCI-N87	Gastric cancer	B-NDG	Succeed	CD3-HER2, ADC+PD-1	Yes	NO
U87-MG	Glioblastoma	B-NDG	Succeed	PD-1 (Keytruda)	22.4%	NO
Hep G2	Hepatocellular carcer	B-NDG	Succeed	PD-1 (Keytruda)	NO	NO

Construction and Application of GvHD Mouse Model

B-NDG mice received irradiation before being engrafted i.v. with PBMC from healthy donors on Day 0. The animals were checked and scored twice weekly for GvHD symptoms including body weight loss, posture, activity, fur texture, and skin damage. The most relevant readout of this model is the survival of the animals. Dead mice received a score of 10 until the end of the experiment.

Assessment of GVHD Model

Induction and Assessment of GvHD in B-NDG Mice.

• B-NDG mice：8-week-old female, Donor1 n=7, Donor2 n=8；
• Preconditioning：1.0 Gy irradiation
• PBMC: i.v.

Clinical scoring criteria for the assessment of GvHD in humanized B-NDG mice.

Criteria	Grade 0	Grade 1	Grade 2
Weight loss	<10%	10%-20%	>20%
Posture	Normal	Hunching noted only at rest	Severe hunching impairs movement
Activity	Normal	Mild to moderate decrease	Severer decrease; no response to any stimulation
Fur texture	Normal	Mild to moderate ruffling/hair loss	Severe ruffling and hair loss
Skin integrity	Normal	Scaling of paws and/or taile	Obvious areas of denuded skin

GvHD model: Preclinical Efficacy of OX40 Antibody in human PBMC engrafted B-NDG Mice

The study showed OX40 antibody improved survival as compared to vehicle.

Preclinical Efficacy of OX40 Antibodies in human PBMC engrafted B-NDG Mice.

• B-NDG mice: 6-week-old female, n=6
• Preconditioning: a 1.0 Gy irradiation
• PBMC: i.v.
• Test article: saline or OX40 antibody (10 mg/kg), i.v., once a week.

Construction and Application of CD34+ HSC Reconstitution Mouse Model

	New-born mice（24h-72h）	Adult mice（4-6w）
CD34+ HSC cells needed	1x104 ~ 1x105	1x105 ~ 2x105
Injection method	Facial vein	i.v. injection
Irradiation	1.0 Gy	1.0-1.2 Gy
Reconstitution period	Long(3–4 weeks)	Long(3–4 weeks)
Costs	$	$$$

Human CD34+ HSC immune reconstitution based B-NDG mice vs B-NDG hIL15 mice

Values are expressed as mean ± SEM.

Values are expressed as mean ± SEM.

huHSC-mice +CDX / PDX model

Cancer Type	Model Type
	huHSC B-NDG hIL15 mice	huHSC B-NDG mice
Breast cancer	MDA-MB-231	MDA-MB-231
Colon carcinoma	RKO, HCT116	RKO
Gastric carcinoma	NUGC-4、SNU620
Lung cancer	A549, A549-hCLDN 18.2, HCC827
Myeloma	NCI-H929
Lymphoma	Daudi、Raji-luc	Raji-luc
Melanoma	A375
Human leukemia cells	K562、HL-60、MV-4-11	K562、MV-4-11
Ovarian cancer		SK-OV-3
Pancreatic cancer	BP0160、BP0209、BP0144

Cell lines for which CDX modeling has been completed, and more cell line modeling is underway.

In vitro cytotoxicity assay of isolated NK cells from huHSC-B-NDG hIL15 mice

Isolated NK cells from the spleen of CD34⁺ HSC immune reconstruction mice (huHSC-B-NDG hIL15 mice, Female, 14-15W) were subjected to in vitro cytotoxicity assay, which showed that the isolated NK cells possess tumor-killing ability against Jurkat cells.

Drug efficacy evaluation in CD34+ HSC reconstituted B-NDG hIL15 mice

B-NDG hIL15 mice reconstituted with CD34+ cells were used for drug efficacy studies.
huHSC-B-NDG hIL15 mice (female, 12-13-week old)
Gastric carcinoma (s.c.)
Test article: Antibody
Values are expressed as mean ± SEM.

CD3-CD20 BsAb anti-tumor efficacy using huHSC-B-NDG hIL15 mice plus

Antitumor activity of CD3×CD20 BsAb in huHSC-B-NDG hIL15 mice plus. Raji cells were subcutaneously implanted into in huHSC-B-NDG hIL15 mice plus (female, 12-week-old, n=6). Mice were grouped when tumor volume reached approximately 80-100 mm3, at which time they were intraperitoneally injected with antibody indicated in table. Values are expressed as mean ± SEM.

In vivo efficacy evaluation of ADC-HER2+Keytruda in huHSC-B-NDG hIL5 mice

B-NDG hIL15 mice reconstituted with CD34+ cells were used for drug efficacy studies.

• huHSC-B-NDG hIL15 mice (female, 16-week old)
• Gastric carcinoma: NCI-N87 (1E7, s.c.)
• Test article: RC48+Keytruda
• Values are expressed as mean ± SEM.