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Microbes & Immunity Establishment of a novel anti-human CCR8 monoclonal antibody

3.2. Flow cytometric analysis The receptors transmit signals to intracellular molecules

Flow cytometric analysis was conducted using purified regarding extracellular conditions and govern broad
C Mab-21 (Figure S1) and commercially available anti- cellular dynamics, such as proliferation, homeostasis,
41,43
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human CD198 (CCR8) mAbs (clone S19017D and L263G8) migration, and motility of the cells. Although the
against CHO-K1, CHO/hCCR8, TALL-1, CCRF-HSB2, therapeutic drugs including mAbs have been developed,
and NK cells. The results showed that C Mab-21, significant challenges remain. These challenges include
8
S19017D, and L263G8 recognized CHO/hCCR8 in a the complexity of the structure, the small area of epitope
dose-dependent manner (Figure 2A). Neither C Mab-21 regions, and the difficulty in purifying these protein as
8
nor L263G8 reacted with parental CHO-K1 cells, even at immunogens. 42,44
a concentration of 20 μg/mL. However, S19017D showed Unlike protein purifications, the preparation of
slight reactivity with CHO-K1 cells at concentrations of antigens using the CBIS method is relatively simple.
20 μg/mL and 2 μg/mL (Figure 2B). For endogenously Furthermore, the CBIS method allows for the retention
hCCR8-expressing cells, C Mab-21 recognized TALL-1, of the antigens’ structure, including modifications such as
8
CCRF-HSB2, and NK cells at concentrations of 2 μg/mL glycosylation and folding. We have successfully developed
and 20 μg/mL (Figure 3). S19017D reacted with TALL-1 multiple mAbs using the CBIS method, targeting human
and CCRF-HSB2 in a dose-dependent manner, even at a epidermal growth factor receptor 1 (HER1; EGFR),
45
concentration of 0.02 μg/mL, but did not recognize NK cells, HER3, trophoblast cell surface antigen 2, CD44, and
47
46
48
even at a concentration of 20 μg/mL (Figure 3). L263G8 podoplanin. Furthermore, some of the mAbs developed
49
reacted with TALL-1 and CCRF-HSB2 at concentration as by the CBIS method exhibited cancer specificity by
low as 0.02 μg/mL, and with NK cells at concentrations of recognizing unique cancer-specific epitopes. 50,51 Therefore,
2 μg/mL or higher (Figure 3). Thus, C Mab-21 could detect the CBIS method is one of the efficient and useful tactics
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both exogenously and endogenously expressed hCCR8 in for generating diverse antibodies targeting membrane
its native conformation using flow cytometry. proteins. Further investigations are required to determine
the epitope of C Mab-21.
3.3. Titration of anti-CCR-8 mAbs on hCCR8- 8
+
overexpressed and endogenously hCCR8- In the immunosuppressive TME, CD8 T cells are
+
expressing cell lines exhausted along with the induction of CCR8 Treg. The
+
infiltration of CCR8 Treg has been shown to associate with
The titration of C Mab-21, S19017D, and L263G8 high thymocyte selection associated high mobility group
8
was assessed with exogenously hCCR8-expressed box (TOX), an exhaustion marker in CD8 T cells, in some
+
CHO/hCCR8 using flow cytometry. The results showed types of cancer patients. 52,53 Elimination of CCR8 Treg
+
that the EC values of C Mab-21, S19017D, and L263G8 for using antibodies is expected to advance the treatment of
50
8
CHO/hCCR8 are 6.5 × 10 M, 2.6 × 10 M, and 1.2 × 10 these cancer patients. Targeting CCR8 may offer more
−8
−9
−9
M, respectively (Figure 4). The histogram of C Mab-21 is specific antitumor activity than other approaches aimed at
8
shown in Figure S2. These results indicate that C Mab-21 Treg removal. 53,54 In mice, CCR8 T cell depletion therapy
+
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possesses a moderate affinity for CHO/hCCR8 cells.
using anti-CCR8 mAbs induces tumor-specific immune
The titration of C Mab-21, S19017D, and L263G8 responses without triggering autoimmune responses
8
was analyzed with endogenously hCCR8-expressing or immune reactions in the TME. Since C Mab-21
36
8
TALL-1 using flow cytometry. The EC values of C Mab- recognizes cell surface hCCR8, we plan to investigate
50
8
21, S19017D, and L263G8 for TALL-1 are 2.0 × 10 M, its potential function against Treg, such as detection
−8
4.6 × 10 −10 M, and 7.8 × 10 M, respectively (Figure 5). and interfering effects, in future studies. Furthermore,
−11
These results showed that C Mab-21 possesses a moderate antibody-dependent cellular cytotoxicity (ADCC) activity
8
affinity for endogenously expressed hCCR8 in TALL-1 and complement-dependent cytotoxicity have previously
leukemia cells. been enhanced by modifying isotypes and defucosylating
Further investigation was conducted to explore other mAbs. 55,56 C Mab-21 is a mouse IgM, which lacks ADCC
8
applications, such as immunohistochemistry. However, activity. Although the crosslinking property of IgM is lost,
hCCR8 could not be detected by immunohistochemistry it will be converted into a mouse IgG version to evaluate
2a
using cell blocks of CHO/hCCR8 (Figure S3). the effect of antitumor activities in xenograft models. We
successfully cloned and determined the complementarity-
4. Discussion determining regions of C Mab-21 (Figure S4).
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Chemokine receptors are focused as targets for many Interestingly, a correlation between cancer-associated
diseases, including inflammatory disorders and cancers. 41,42 fibroblasts (CAFs) and CCR8 has been found from
Volume 2 Issue 2 (2025) 129 doi: 10.36922/mi.4661