Longitudinal analysis of T-cell phenotype and function during bispecific antibody therapy Free

Introduction

Bispecific antibodies (BsAbs) linking CD3 on T cells to CD20 on lymphoma cells (CD3xCD20) or to BCMA on myeloma cells (CD3xBCMA) are a significant advancement in the treatment of hematologic malignancies. However, most patients do not have durable responses. While BsAbs offer an “off-the-shelf” immunotherapeutic approach, their efficacy depends on the functionality of the patient's T-cell compartment, which is often compromised by prior chemotherapy and the immunosuppressive tumor microenvironment (TME). Prior studies have reported elevated inhibitory markers and reduced activation (e.g., interferon release) in both lymphoma and myeloma. However, no studies have systematically evaluated T-cell phenotype and function during continuous BsAb treatment. Most existing data focus on pre-treatment or relapse timepoints. Little is known about longitudinal phenotypic changes and the capacity of patient T cells to respond to viral antigens during therapy.

Methods

Patient samples were collected under an IRB-approved protocol at baseline, 1 week, 1 month, and every 3 months until relapse. PBMCs were isolated via density gradient centrifugation and analyzed for T-cell phenotype and virus-specific T-cell (VST) function. Flow cytometry was used to assess lineage markers (CD3, CD4, CD8, CD19, CD56, CD14), differentiation states (naïve, central memory, effector memory, terminally differentiated via CD45RO/RA, CCR7, CD62L, CD28), and exhaustion markers (PD-1, CTLA-4, TIM-3, LAG-3, CD39). ELISPOT assays were performed following stimulation with EBV, CMV, adenovirus, and BK virus antigens to quantify IFN-γ–secreting VSTs. To date we collected samples from 7 patients pre and post BsAb treatment, with a range of 1-8 collection time points.

Results

We analyzed longitudinal immune and VST profiles in six BsAb-treated patients (CD3xBCMA: n=1; CD3xCD20: n=5), with timepoints up to 13 months post-treatment. PD-1 expression was elevated at baseline (mean 22.4%, range 8.6-31.6%) and remained high throughout treatment (mean 20.0%, mean 8.9-40.6%), while other exhaustion markers (TIM-3, LAG-3, CTLA-4) were largely absent. Longitudinal flow cytometry revealed a trend toward decreased naïve T cells (TN) post-treatment. Central memory T cells (TCM) declined in three of four patients, while effector memory subsets (TEMRA and TEM) increased, suggesting a shift toward differentiation during therapy.

CD14⁺CD56⁺ monocytes, considered an atypical, proinflammatory phenotype, were elevated in 5/6 patients prior to treatment compared to healthy donors, suggesting a pre-existing dysregulated immune environment. In 2 patients levels declined post-treatment, 2 patients levels fluctuated with ongoing treatment, and one remained elevated after treatment.

CMV-specific T cells (CMVSTs) dominated the VST landscape across all patients. EBV-, BKV-, and AdV-specific responses were sporadic and generally low. Healthy donors also showed CMV dominance but had broader VST diversity. Patients with ongoing responses (n=2) showed minimal change in CMVSTs (mean change: –3.0%), while those who relapsed (n=2) exhibited a decline (mean change: –66.6%).

Conclusion

In our study, patients treated with continuous BsAb had elevated PD-1 expression at baseline, which persisted throughout treatment. Interestingly, other exhaustion markers were largely absent. CD14⁺CD56⁺ monocytes, an atypical and proinflammatory phenotype, were elevated in 5 out of 6 patients prior to treatment, suggesting a pre-existing dysregulated immune environment.

Most patients experienced relapse, raising the possibility that baseline T-cell exhaustion and/or an inflammatory microenvironment may have contributed to the lack of durable responses. Treatment was associated with increased T-cell differentiation, accompanied with a decline in naïve and central memory subsets. As TN and TCM subsets respond to new and previously encountered pathogens, their loss may increase the risk of infection in BsAb treated patients. Virus-specific T-cell responses, particularly against CMV, were preserved in clinical responders, but declined immediately prior to or at the time of relapsed, suggesting that antiviral T-cell function may serve as a surrogate for T-cell fitness and treatment durability. These findings support the utility of longitudinal immune monitoring to identify correlates of response and resistance to BsAb therapy.