hCD7(V2)

Fig.1 Detection of CD7 expression in duodenum and small intestine by RT-PCR. 

Wild type: only one band at 107 bp with primers F1/R1 (mCd7);

Homozygous: only one band at 163 bp with primers F2/R2 (hCD7).

Abbr. M, DNA marker; HO, homozygous; WT, wild type.

Fig.2 Detection of m/h CD7 expression on T cells in Blood (A), spleen (B) and thymus (C) in hCD7(2) KI mice.

Fig.3 Detection of hCD7 expression on T cells in Blood (A), spleen (B) and thymus (C) in hCD7(2) KI mice.

Fig.4 Detection of m/h CD7 expression on NKs in Blood (A) and spleen (B) in hCD7(2) KI mice.

Fig.5 Analysis of leukocytes cell subpopulation in Spleen (A) and thymus (B) in WT and HO hCD7(2) mice.

Fig.6 Body weight and body weight change among all groups. HE hCD7(V2) mice (18 weeks, male) received a single intravenous injection of LNP control (10 µg), anti-hCD7 VHH-LNP (10 or 25 µg). Body weight was measured at baseline (Day 0, prior to injection) and at 24 hours post-injection (Day 1, prior to sacrifice). (A) Absolute body weight at Day 0 and Day 1. (B) Percentage of body weight change from baseline. (n=1/group)

Abbr. LNP, lipid nanoparticle; VHH, variable domain of heavy chain-only antibody; HE, homozygous; WT, wild-type; i.v., intravenous

Fig.7 In vivo bioluminescence imaging of hCD7-targeted LNPs in heterozygous hCD7(V2) mice. HE hCD7(V2) mice were intravenously administered with either 10 µg or 25 µg of CD7 VHH‑conjugated LNP encapsulating luciferase‑encoding mCherry mRNA. (A)Whole body image from dorsal and ventral side were imaged one day post‑injection. (B)Organs including brain, heart, liver, spleen, lung, kidney, and lymph nodes (LN) were harvested , anafter whole body image. luciferase expression was assessed by IVIS. Representative bioluminescence signals (in relative radiance, e.g., photons/sec/cm²/sr) are shown for each organ across different dose groups. 

 In HE hCD7(V2) mice, the anti-hCD7VHH-LNP demonstrated great targeting ability to the liver, spleen and LN in a dose-depedent manner. 

Fig.8 Quantitative bioluminescence analysis of hCD7-targeted LNPs in heterozygous hCD7(V2) mice. HE hCD7(V2) mice were intravenously administered control LNP or CD7VHH-conjugated LNP at 10 µg or 25 µg. One day post-injection, luciferase expression from (A) whole body from dorsal and ventral side and (B) harvested organs was assessed by IVIS. Bar graphs show quantified radiance (photons/sec/cm²/sr) for each organ across all groups.

The anti-hCD7VHH-LNP showed dose-dependent targeting in the spleen. 

Fig.9 mCherry expression on lymphocyte subpopulation in the spleen of HE hCD7 mice by FACS. Splenocytes were collected from HE hCD7(V2) mice (male, 18 weeks old) adminstrated with 10 or 25 µg LNP,i.v., and analyzed one day later by flow cytometry.

FACS analysis of splenocytes from HE hCD7(V2) mice revealed that mCherry expression was prominently detected on NK cells, whereas CD3+ T cells exhibited relatively minimal mCherry expression, likely due to lower CD7 surface expression on T cells compared to NK cells. Accordingly, surface CD7 expression levels on both NK and T cells were reduced, indicating specific recognition of CD7-positive cells by the anti-CD7 VHH-LNP.