•  CD3 Molecule: As a vital component of the T-cell receptor (TCR) complex, CD3 is responsible for transducing antigen recognition signals into the cell interior. The engagement and ligation of CD3 can directly induce T-cell activation.
•  CD8 Molecule: CD8 Binding of MHC-peptide complexes in cis or trans Regulates CD8+ T-cell Responses. The coreceptor CD8αβ can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation.
• T-Cell Engagement (TCE): CD3 redirection bispecific antibodies bind tumor associated antigens (TAA) on cancer cells and CD3 on recruited T cells to elicit cancer cell cytotoxicity independent of T cell receptor specificity. Cancer cell cytotoxicity occurs through perforin and granzyme B release resulting in apoptosis through caspase pathway induction.
• CD3/CD8/TAA Antibody: To circumvent the unintended activation of regulatory T cells (Tregs), which are typically CD4+, the antibody is engineered with a preferential affinity for CD8+ cells. This selectivity enhances cytotoxic efficacy while simultaneously mitigating immunosuppressive effects.

CD3EDG

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hCD8 mice.

CD8

• The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8a gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8A expression is driven by endogenous mouse CD8a promoter, while mouse Cd8a gene transcription and translation will be disrupted.
• The exons 1-3 and partial exon 4 of mouse Cd8b1 gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8b1 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8B1 expression is driven by endogenous mouse CD8b1 promoter, while mouse Cd8b1 gene transcription and translation will be disrupted.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426).

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood was collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392).

• The frequencies of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hCD3EDG/hCD8 mice were similar to those in C57BL/6JNifdc mice
• Humanization of CD3EDG and CD8 does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes, and thymus were isolated from C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hCD3EDG/hCD8 mice were comparable to those in C57BL/6JNifdc mice
• Humanization of CD3EDG and CD8 does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes, and thymus were isolated from C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• B-hCD3EDG/hCD8 mice have normal T cell immunogenic function induced by the OVA.

Detection of OVA-induced immune responses in B-hCD3EDG/hCD8 mice by IFN-γ ELISpot assay. Male wild-type C57BL/6JNifdc mice and B-hCD3EDG/hCD8 mice at the age of 9–10 weeks were immunized with intraperitoneal injection of 0.3 mg of OVA protein (Simga, A5503-25MG) and 30 μg poly (I:C) (InvivoGen, tlrl-pic). Mice were immunized with OVA two times at 1-week interval. One week after the last immunization, mice were sacrificed. The splenocytes were extracted, stimulated with OVA peptide257–264, or Cell Activation Cocktail (without Brefeldin A), (BioLegend, 42330) as positive control (PC), and then measured for IFN-γ secretion. (A) Representative results showing stimulation of splenocytes harvested from immunized mice with OVA peptide257–264, or positive control in duplicates. (B) Summary of results. These data indicate that B-hCD3EDG/hCD8 mice have normal T cell immunogenic function.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

In vitro cytokine production (IFN-γ and IL-2) in B-hCD3EDG/hCD8 mice. T cells (2×10⁵) were isolated from the splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), incubated in the presence of anti-mCD3ε antibody (2 ug/ml，BioXcell，BE0001-2) or anti-hCD3ε antibody (2 ug/ml，BioXcell，BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method.

In vitro cytotoxicity evaluation of a CD3/EPCAM bispecific antibody against human EPCAM-expressing MC38 cells. B-hEPCAM MC38 cells were labeled with a violet cell tracker fluorescent dye and seeded into 96-well plates. On the following day, splenocytes isolated from B-hCD3EDG/hCD8 mice were added and cocultured with the target cells in the presence of various concentrations of the CD3/EPCAM bispecific antibody (Solitomab analog, commercial). Cytotoxicity and the proportion of CD4+ T / CD8+ T cell activation was assessed after 48 hours of incubation. Effector-to-target (E:T) cell ratios of 40:1 were used.

Establishment of a B-hEPCAM MC38 model and in vivo efficacy study of an anti-human CD3/EPCAM bispecific antibody. B-hEPCAM MC38 colon cancer cells were implanted subcutaneously into homozygous B-hCD3EDG/hCD8 mice (male, 7-week-old, n=5). When the average tumor volume reached approximately 110 mm³, mice were randomized and subsequently administered the anti-human CD3/EPCAM bispecific antibody (Solitomab analog, commercial) via intravenous injection.

Antitumor activity of anti-human CD3/EPCAM bispecific antibody in B-hCD3EDG/hCD8 mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, CD3/EPCAM bispecific antibody (Solitomab analog, commercial) was efficacious in controlling tumor growth in B-hCD3EDG/hCD8 mice, demonstrating that the B-hCD3EDG/hCD8 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD3-based TCE bi/multispecific antibodies. Values are expressed as mean ± SEM.

• The proportions of CTL cells in the blood increased in the CD3/EPCAM BsAb treatment group.
• The proportions of CTL cells and Th cells in the spleen and tumor remained unchanged in the CD3/EPCAM BsAb treatment group.

The proportions of mCD69+ CTL cells and mGranzyme B+ CTL cells in the spleen were increased in CD3/EPCAM BsAb treatment group.

The proportions of mCD25+ CTL cells and mGranzyme B+ CTL cells in the blood were increased in CD3/EPCAM BsAb treatment group.

The proportions of mCD69+ CTL cells, mCD25+ CTL cells and mGranzyme B+ CTL cells in the tumor were increased, and the proportions of mKi67+ CTL cells and mKi67+ Th cells were decreased in CD3/EPCAM BsAb treatment group.