CD3×CD19 Bispecific Antibody-Based Therapy

• Gene Information: CD3: Encoded by CD3E, D, G; part of the Ig superfamily. It forms the essential signaling backbone of the T-cell receptor (TCR) complex. CD19: is a transmembrane glycoprotein, located on chromosome 16p11.2. It belongs to the immunoglobulin superfamily and plays a critical role in B-cell development, activation, and maintenance throughout the immune system.
• Protein Expression: CD3: Constitutive and universal marker for all mature T cells (CD4+, CD8+). Always present on the cell surface. 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.
• Signaling Pathway: CD3: Operates via ITAM phosphorylation and ZAP-70. It triggers the initial "on" switch, Ca2+ flux, and immediate cytotoxicity. CD19: a key co-receptor of the B-cell receptor (BCR) complex. By interacting with molecules such as CD21 and CD81, it amplifies BCR-mediated signaling and activates downstream pathways including PI3K/AKT and MAPK/ERK.
• Therapeutic Inhibition: CD3×CD19 bispecific antibodies recruit and activate T cells by simultaneously binding CD3 on T cells and CD19 on B cells, leading to targeted killing of CD19-positive malignant cells. This approach has demonstrated strong clinical efficacy in B-cell malignancies, with Blinatumomab being the first approved CD3×CD19 bispecific antibody. Ongoing efforts aim to improve efficacy, durability, and safety.

• Mouse CD3E was detected on T cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/hCD19 ad mice.
• Human CD3E was detected on T cells populations in B-hCD3EDG/hCD19 ad mice, but not in wild-type C57BL/6 mice.

Mouse and human CD3E expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19 ad mice (female, 8-week-old, n = 1). CD3E expression on T cells was analyzed by flow cytometry using species-specific anti-CD3E antibodies (anti-human CD3E antibody, BD Horizon, 562426; anti-mouse CD3E antibody, Biolegend, 100312 ).

• Mouse CD19 was detected on B cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/hCD19 ad mice.
• Human CD19 was detected on B cells populations in B-hCD3EDG/hCD19 ad mice, but not in wild-type C57BL/6 mice.

Mouse and human CD19 expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 and homozygous B-hCD3EDG/hCD19 ad mice (female, 8-week-old, n = 1). CD19 expression on B cells was analyzed by flow cytometry using species-specific anti-CD19 antibodies (anti-human CD19 antibody, BD Horizon, 302208; anti-mouse CD19 antibody, Biolegend, 115538 ).

In vivo B cell depletion of anti-human CD3/CD19 bispecific antibody (BsAb) in homozygous B-hCD3EDG/hCD19 ad mice. Anti–human CD3/CD19 bispecific antibody and PBS were administered into B-hCD3EDG/hCD19 ad mice (G1:n=2; G2: n=3) through a single dose injection. Blood  were collected at Day-4, Day1, and Day7 after treatment. The numbers and the frequency of mCD45+ cells, B cells (mB220+) and T cells (mTCRβ+) were determined by flow cytometry. (A) The number of CD45+ cells, B cells and T cells in blood. (B) The proportion of T and B cells in the CD45+Ly6G- cells. Values are expressed as mean ± SEM.