CD3/Lymphocytes in Tumor RejectionCD8/TAA MOA: Specifically Enhancing the Role of CD8+ Cytotoxic

• Gene Information: 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. 
• CD8-guided T-cell Engagers: 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. The antibody targeting CD3/CD8/TAA is engineered with a preferential affinity for CD8+ T cells. This selectivity enhances cytotoxic efficacy while simultaneously mitigating immunosuppressive effects.

Gene Information: CD19: is a transmembrane glycoprotein. It belongs to the immunoglobulin superfamily and plays a critical role in B-cell development, activation, and maintenance throughout the immune system. BCMA: Encoded by the TNFRSF17 gene, a critical member of the tumor necrosis factor receptor (TNFR) superfamily. 

Protein Expression: CD19: is expressed almost exclusively on B-lineage cells, beginning at the early B-cell stage and persisting through mature B cells. Its expression is typically absent or markedly reduced in plasma cells. BCMA: It is a B-cell maturation antigen, whose expression is highly in mature plasma cells and nearly all multiple myeloma cells.

• Mouse CD3E was detected in T cells of wild-type C57BL/6JNifdc mice, but not in B-hCD3EDG/hCD8/hBCMA/hCD19 ad Mice. 
• Human CD3E was exclusively detected in T cells of B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized 1mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized mice by flow cytometry. Splenocytes and blood cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized Mice, and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100210) and anti-human CD3ε antibody (Biolegend, 317344).

• Human CD8a and CD8b were exclusively detectable in TCRβ+ and CD4-T cells from homozygous B-hCD3EDG/hCD8/hBCMA/ 
• hCD19 ad mice, but not in wild-type C57BL/6JNifdc mice.

CD8 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice by flow cytometry. 

Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice (male, 7-week-old). Protein expression was analyzed with anti-mouse CD8 antibody (Biolegend, 100730) anti-human CD8a antibody (Biolegend, 300908) and anti-human CD8b antibody (BD, 742392) by flow cytometry. 

• Human CD8a and CD8b were exclusively detectable in NKT cells from homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice, but not in wild-type C57BL/6JNifdc mice.

CD8 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice by flow cytometry. 

Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice (male, 7-week-old). Protein expression was analyzed with anti-mouse CD8 antibody (Biolegend, 100730) anti-human CD8a antibody (Biolegend, 300908) and anti-human CD8b antibody (BD, 742392) by flow cytometry. 

• Human CD8a and CD8b were exclusively detectable in NKT cells, T cells and CD4-T cells from homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice. 
• Mouse CD8a were not detected in B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice.

Summary of CD8 expression profile in homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanzied mice. 

• Human BCMA was detectable in plasma cells of spleen from B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized mice but not in wild-type C57BL/6JNifdc mice.

Human BCMA expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad huamanized mice (male, n=3). Human BCMA expression on plasma cells was analyzed by flow cytometry using anti-human BCMA antibody (Biolegend, 357504). Mice were stimulated with γ-secretase inhibitor to enhance membrane BCMA expression.

• Human CD19 was exclusively detectable in homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized mice, but not in wild-type C57BL/6JNifdc mice.

CD19 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8/hBCMA/hCD19 ad mice by flow cytometry. Splenocytes and blood were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hBCMA/hCD19 ad humanized mice (male, 7-week-old). Protein expression was analyzed with anti-mouse CD19 antibody (Biolegend, 115507) and anti-human CD19 antibody (Biolegend, 302234) by flow cytometry.