Heterodimeric Bispecific Antibodies Direct T Cell-Mediated Cytotoxicity Against Desmoplastic Small Round Cell Tumors 

Background

Memorial Sloan Kettering is a cancer treatment and research center in New York City. During the summer of 2024, I worked in the Cheung Lab to evaluate the effectiveness of antibody-based immunotherapies against various forms of cancer. 

Desmoplastic Small Round Cell Tumors (DSRCT) are soft tissue sarcomas that grow in the abdomen/pelvic area. DSRCT could be treated with bispecific antibodies (BsAbs), which redirect T cells to kill the tumor. DSRCT cells highly express CD24 and HER2, and targeting either protein using BsAbs has been shown to have anti-tumor efficacy. However, targeting only one molecule can lead to toxicity against healthy tissues with CD24 or HER2 expression. 

Therefore, using a heterodimeric BsAb to target CD24 and HER2 (CD24 x HER2) simultaneously may reduce toxicity since DSRCT is the only type of tissue with a high expression of both proteins. However, we also needed to assess whether CD24 x HER2 can maintain the same efficacy as homodimeric BsAbs targeting only one protein (CD24 x CD24, HER2 x HER2). 

Aim

Compare the efficacy of the CD24 x HER2 heterodimeric BsAb against homodimeric BsAbs when performing immunotherapy against DSRCT in vitro and in vivo. 

Hypothesis

CD24 x HER2 will retain the same anti-DSRCT efficacy as homodimeric BsAbs.

Experimental Groups

We evaluated 6 different BsAbs that engaged 3 targets - CD24, HER2, and CD33. CD33 is a negative control target because it is not found on DSRCT. The BsAbs are as follows:

• CD33 x CD33: Negative control

• CD24 x CD33: Monovalent control (only CD24 arm binds to DSRCT)

• HER2 x CD33: Monovalent control (only HER2 arm binds to DSRCT

• CD24 x CD24: Positive control

• HER2 x HER2: Positive control

• CD24 x HER2: Experimental group

Methods

Chromium release assays were performed on JN-DSRCT-1 and BOD DSRCT cell lines. Next, 6 groups of 7 mice bearing DSRCT xenografts were each treated with human T cells and one BsAb. On Day 4, tumors were harvested from 2 mice from each group to perform immunohistochemistry for T cell localization. The remaining 5 mice were treated with BsAbs 2x/week as well as T cells and IL-15 1x/week. Their tumors were measured 2x/week and the mice were weighed 1x/week.

Chromium Release Assays

• CD24 x HER2 has the lowest or second-lowest EC50 (concentration where 50% killing occurs). 

• CD24 x HER2 has the highest or second-highest maximum killing (killing percentage at maximum concentration of BsAb). 

Both of these metrics indicate that CD24 x HER2 retains the same efficacy as homodimeric BsAbs. 

IHC

CD24 and HER2 BsAbs induce T cell trafficking to BER xenografts. 

Tumor Volume Graph 

All groups had a dramatic decrease in tumor volume except for CD33 x CD33, the negative control. Even the monovalent controls showed a strong response. This indicates efficacy between the 5 groups is indistinguishable. 

Tumor volume is still being monitored to check for recurrences and to determine whether there are differences between monovalent and bivalent groups. 

Weight Graph

The mice are retaining the same weight, meaning there is no obvious toxicity. This is expected since the T cells are human and hence only attack human cells, while leaving the rest of the mouse alone. 

Conclusion

CD24 x HER2 retains the same anti-DSRCT efficacy of CD24 and HER2 homodimeric BsAbs.

Future Directions

The next step of our study is evaluating whether CD24 x HER2 reduces toxicity compared to homodimeric BsAbs targeting CD24 and HER2. This would be done by testing the same panel of BsAbs against single-target knockdown DSRCT cell lines (e.g., DSRCT with only CD24 or only HER2), which are surrogates for normal human tissues with CD24 or HER2 expression. In theory, CD24 x HER2 should be less effective against these knockdown cell lines than homodimeric BsAbs.