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動物の臨床観察

全血球計算

血液の化学解析

組織病理学(ホルマリン固定パラフィン包埋(FFPE)検体、H&EIHC)

毒性メカニズム解析

事例1:B-hCD40マウスを利用した抗ヒトCD40の有効性と安全性の評価

1. B-hCD40マウスで抗ヒトCD40抗体のin vivo効果と安全性の評価

抗人CD40的功效

Murine colon cancer MC38 cells were subcutaneously implanted in homozygous B-hCD40 mice, where the extracellular domain of human CD40 replaces that of mouse CD40 via genomic knock-in. Mice were grouped when the average tumor size reached approximately 150±50 mm3 (n=5). Selicrelumab significantly inhibited tumor growth, confirming that the B-hCD40 mouse model is a powerful tool for in vivo studies of anti-human CD40 antibodies in mice. Tumor volume: mean ± SEM (A).  Body weight: mean ± SEM (B)

2. B-hCD40マウスで抗ヒトCD40毒性の包括的な評価(CBC、血液化学、サイトカインプロファイリング、および免疫細胞プロファイリング(フローサイトメトリー)

A. 抗ヒトCD40投薬計画及び治療中の体重の変化

评估反人类

Anti-human CD40 antibodies were administered i.p. in homozygous B-hCD40 mice on days indicated by red arrows (n=5). No significant difference in average body weight was detected over the course of the treatment.


B. 抗ヒトCD40抗体で処理の後B-hCD40マウスの全血球数値(CBC)

全血细胞计数

AbbreviationStand forStand forStand for
WBCWhite Blood Cell CountMCVMean Corpuscular Volume
NE#Neutrophil CountMCHMean Corpuscular Hemoglobin
LY#Lymphocyte CountMCHCMean Corpuscular Hemoglobin Concentration
MO#Monocyte CountMPVMean Platelet Volume
RBCRed Blood Cell CountPLTPlatelet Count
HbHemoglobinRDWRed Cell Distribution Width
HCTHematocrit

CBC was performed with anti-CD40 antibodies in B-hCD40 mice two days after the termination of efficacy evaluation experiment.



C. 抗ヒトCD40抗体で処理したB-hCD40マウスの血液生化学検査

抗人血液化学

AbbreviationStands forExplanation
ALTAlanine transaminaseHigh level indicates liver injured
ASTAspartate transaminaseHigh level indicates liver damage
CHOLCholesterolHigh level indicates high blood liquid
CRCreatinineHigh level indicates low glomerular filtration
GLUGlucoseHyperglycemia or hypoglycemia
TRIGTriacylglycerolHigh level indicates high blood lipid
UREAUreaHigh level indicates kidney damage, liver disease, diabetes or infection

The blood chemistry was tested with anti-human CD40 antibodies in B-hCD40 mice two days after the termination of efficacy evaluation experiment.


D. 抗ヒCD40抗体で処理したB-hCD40マウスのサイトカインの測定


Cytokine profiles in anti-human CD40 antibody treated MC38-bearing B-hCD40 mice. Cytokines were measured by LEGENDplexTM (Biolegend). Anti-human CD40 Ab4 and Ab5 showed different cytokine profiles. Anti-human CD40 Ab4 treatment led to significant increase several cytokines including IFN-γ, MCP-1, and IL-27. Anti-human CD40 Ab5 resulted in significant increase of IL-10. Both antibodies modestly elevated TNF-α.

细胞因子分析


E. B-hCD40マウスに抗ヒトCD40抗体投与の後免疫細胞分析

免疫细胞分析

Flow cytometry was performed to examine immune cell profiles after anti-human CD40 antibody treatment in B-hCD40 mice. The results indicated that CD40 Ab4 treatment resulted in a significant increase in the percentage of activated CD4 and CD8 T cells and B cells.


F. B-hCD40マウスに抗ヒトCD40抗体投与によるB細胞活性化

B淋巴细胞活化

Anti-human CD40 antibodies were administered i.p. in homozygous B-hCD40 mice After 48h, abdominal cells and splenocytes were collected and analyzed for expression of activation markers by flow cytometry. Selcrelumab led to significant elevation of activation markers in both spleen and abdomen.


3.B-hPD-1/hCD40マウスに対してで抗ヒトCD40抗体毒性の評価CBC、血液生化学及び組織病理学)

评估抗人CD40

AbbreviationStand forAbbreviationStand for
WBCWhite Blood Cell CountMCVMean Corpuscular Volume
NE#Neutrophil CountMCHMean Corpuscular Hemoglobin
LY#Lymphocyte CountMCHCMean Corpuscular Hemoglobin Concentration
MOMonocyte CountMPVMean Platelet Volume
RBCRed Blood Cell CountPLTPlatelet Count
HbHemoglobinRDWRed Cell Distribution Width
HCTHematocrit

评估抗人CD40细胞

  1. Effect of an anti-human CD40 antibody in CBC test in B-hPD-L1/hCD40 mice, where the extracellular domains of both hPD-L1 and hCD40 replace their respective murine counterpart via genomic knock-in. Antibody treatment 6 days after the initial dose led to decrease of MCV, PLT and LY# and increase of NE#, MPV and RDW.

  2. Body weight profile over the course of after antibody administration. No persistent body weight changes were observed.

  3. AST of B-hPD-1/hCD40 mice treated with higher doses increased significantly compared to vehicle control group. Microscopic changes were observed in B-hPD-1/hCD40 mice from the anti-hCD40 antibody treated group of 30mg/kg.

  4. Elevated infiltration of mixed inflammation cells in the interstitium as shown by H&E staining of liver, liung, pancreas, and intestine.

事例2:B-hCD47/hSIRPAマウスを用いて抗ヒトCD47抗体のin vivo有効性と安全性評価

1. B-hCD47/hSIRPAマウスで抗ヒトCD47抗体のin vivo薬効及び毒性の評価

功效与毒性CD47

Colon cancer MC38-hCD47 cells engineered to express human CD47 were subcutaneously implanted in homozygous B-hCD47/hSIRPA mice, where the extracellular domains of both hCD47 and hCSIPRA replace their respective murine counterpart via genomic knock-in. The mice were grouped as the average tumor volume reached 150±50mm3 (n=5). Anti-human CD47 antibodies demonstrated anti-tumor efficacy in this model. Antibody treatment also resulted in a rapid but moderate body weight loss, which was recovered by day 10. These results indicate that B-hCD47/hSIRPA mice are a useful model for testing anti-human hCD47 antibody efficacy and observing whole body toxicity. Data is expressed as mean ± SEM of tumor volume and body weight.


2. B-hCD47/hSIRPAマウスで抗ヒトCD47抗体の血液及び肝臓毒性に対すの評価

A. 抗ヒトCD47抗体の血液毒性特性

血液学毒性指示

Indication of hematological toxicity of Anti-hCD47 antibodies administered i.p. in homozygous B-hCD47/hSIRPA mice. Blood were collected two days after the treatment. Significant decrease of red blood cell counts (RBC), hematocrit (HCT),  were observed upon anti-human CD47 treatment. Monocyte counts were decreased. Anti-human CD47 Ab2 also led to decrease of platelet count.

AbbreviationStand forStand forStand for
WBCWhite Blood Cell CountMCVMean Corpuscular Volume
NE#Neutrophil CountMCHMean Corpuscular Hemoglobin
LY#Lymphocyte CountMCHCMean Corpuscular Hemoglobin Concentration
MO#Monocyte CountMPVMean Platelet Volume
RBCRed Blood Cell CountPLTPlatelet Count
HbHemoglobinRDWRed Cell Distribution Width
HCTHematocrit

Anti-hCD47 antibodies were i.p. injected into homozygous hSIRPA/hCD47 mice. Blood were collected two days after the treatment and analyzed by Blood Routine Test.


B. 抗ヒトCD47抗体の肝臓び対する毒性

抗人肝毒性

AbbreviationStands forExplanation
ALTAlanine transaminaseElevated level indicates liver damage
ASTAspartate transaminaseElevated level indicates liver damage

Anti-hCD47 antibodies led to liver damage as indicated by elevated levels of liver enzymes in blood in homozygous hSIRPA/hCD47 mice. Blood was collected two days after the initial treatment.

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