Description

• CD98, also known as 4F2, is a widely expressed disulfide-linked heterodimer in the solute transporter family. It consists of an 85 kDa glycosylated type II transmembrane heavy chain and a 40‑50 kDa non-glycosylated light chain with 12 transmembrane segments. It is expressed on T, B, NK cells, granulocytes and many cell lines. The multifunctional protein CD98 heavy chain (CD98HC, Slc3a2) associates with integrin b1 through its cytoplasmic and transmembrane domains and the CD98HC-mediated integrin signaling is required for maintenance of ES cell proliferation. The extracellular domain of CD98HC interacts with L-type amino acid transporters (LATs) and is essential for appropriate cell surface distribution of LATs (SLC7A5, 6, 7, 8, 10, or 11). LATs mediate the transport of amino acids and other molecules such as thyroid hormone.

• The exons 2-10 of mouse Cd98 gene that encode the extracellular domain were replaced by human CD98 exons 4-12 in B-hCD98HC mice.

• Human CD98HC was exclusively detectable on T cells, B cells, and brain microvascular endothelium of homozygous B-hCD98HC mice. Humanization of CD98HC does not change the overall frequency or distribution of immune cell types in spleen, blood, and lymph nodes. CD98HC humanized does not change the blood cell composition and morphology, ALT and AST levels or health of liver.

• This product is used for tumor pharmacology and safety evaluation of anti-CD98HC antibody, as well as assessing the potential of the drug to penetrate the blood-brain barrier.

Targeting strategy

Gene targeting strategy for B-hCD98HC mice. The exons 2-10 of mouse Cd98 gene that encode the extracellular domain were replaced by human CD98 exons 4-12 in B-hCD98HC mice.

mRNA expression analysis

Strain specific analysis of CD98 gene expression in wild type (WT) mice and B-hCD98HC mice by RT-PCR. Mouse Cd98 mRNA was detectable only in kidney of WT mice (+/+). Human CD98 mRNA was detectable only in homozygous B-hCD98HC mice (H/H) but not in WT mice (+/+).

Protein expression analysis in spleen

Strain specific CD98 expression analysis in homozygous B-hCD98HC mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD98HC mice (H/H), and analyzed by flow cytometry with species-specific anti-CD98 antibody. Mouse CD98 was detectable in WT mice (+/+). Human CD98 was exclusively detectable in homozygous B-hCD98HC mice (H/H) but not in WT mice (+/+).

Protein expression analysis in brain

Strain specific CD98 expression analysis in homozygous B-hCD98HC mice by Immunofluorescence staining. Brain was collected from wild type (WT) mice and homozygous B-hCD98HC mice (female,8 week old) and processed into paraffin sections. The paraffin sections were stained using an species-specific anti-human CD98HC antibody (green). Brain tissues were co-stained with an anti-mouse CD31 antibody to visualize microvascular endothelial cells (red). Representative results of WT mice (A) and homozygous B-hCD98HC mice (B) are shown. The results indicated that human CD98 was exclusively detectable on brain microvascular endothelium of homozygous B-hCD98HC mice, but not in WT mice.

Frequency of leukocyte subpopulations in spleen

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD98HC mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD98HC mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD98HC does not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hCD98HC mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD98HC mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD98HC does not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in lymph node

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD98HC mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCD98HC mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD98HC does not change the frequency or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Hematology analysis

Complete blood count (CBC) of B-hCD98HC mice. Values are expressed as mean ± SD.

Biochemistry analysis

Biochemical test of B-hCD98HC mice. Values are expressed as mean ± SD.

In vivo PK evaluation of anti-human CD98HC BsAbs

In vivo pharmacokinetic (PK) evaluation of anti-human CD98HC bispecific antibodies (BsAbs). B-hCD98HC mice were injected with control IgG (10 mpk) and anti-human CD98HC BsAbs (13.3 mpk produced in-house) via tail vein. Brain and serum were taken for in vivo PK evaluation. Brain concentrations(A), serum concentrations (B), and brain-to-serum ratio (C) of anti-human CD98HC BsAbs were quantified. As shown in panel, anti-human CD98HC BsAbs exhibited higher serum clearance and enhanced brain exposure after dose. The results confirmed that brain of B-hCD98HC mice enables uptake of an intravenously administered anti-human CD98HC BsAbs and B-hCD98HC mice provide a powerful preclinical model for in vivo evaluation of effective delivery of protein therapeutics to the central nervous system (CNS). Graphs represent mean ± SEM.

CH98HC Antibody could penetrate brain parenchyma

IHC staining of anti-CD98HC Abs penetrate brain parenchyma. B-hCD98HC mice were injected with control IgG (10 mpk) and anti-human CD98HC Abs (18.8 mpk produced in-house) via tail vein. Brain comparts were taken for IHC staining after 120 h. The results confirmed that brain of B-hCD98HC mice enables uptake of an intravenously administered anti-human CD98HC Abs and B-hCD98HC mice provide a powerful preclinical model for in vivo evaluation of effective delivery of protein therapeutics to the central nervous system (CNS).