Bivalent bispecific CD28 antibodies reinforce T-cell responsiveness and revert anergy/quiescence in patients treated with bispecific CD3 antibodies

This study demonstrates that engineered bivalent bispecific CD28 antibodies (BiCos) effectively reverse T-cell anergy and quiescence induced by CD3-engaging bispecific T-cell engagers (TCEs) by restoring costimulatory signaling, thereby potently enhancing antitumor immunity and enabling tumor elimination even at low TCE doses.

The Gist

The Big Picture: A Broken Engine and a Missing Spark

Imagine your body's immune system as a highly trained army of soldiers (T-cells) designed to hunt down and destroy cancer. For years, doctors have been using a powerful weapon called a T-cell Engager (TCE). Think of a TCE as a "magnet" that grabs a cancer cell with one hand and grabs a T-cell with the other, forcing them to shake hands. This handshake is supposed to wake up the T-cell and tell it, "Attack!"

The Problem:
The researchers discovered a hidden flaw in this strategy. When they used the TCE alone (without any other help), the T-cells would attack briefly, but then they would get tired, confused, and eventually shut down.

In the paper, they call this state "anergy" or "quiescence."

• The Analogy: Imagine you are trying to start a car. You turn the key (Signal 1 from the TCE), and the engine sputters. But you never press the gas pedal. The engine doesn't die, but it refuses to run. The T-cells are stuck in "idle mode." They are alive, but they aren't fighting. They become "hyporesponsive"—basically, they are too polite to do their job.

The Solution: The "Gas Pedal" (BiCos)

The scientists realized that to get the car moving, you need two things:

1. The Key (Signal 1): The TCE that grabs the cancer and the T-cell.
2. The Gas Pedal (Signal 2): A second signal that tells the T-cell, "Now, go! Attack with full force!"

In the past, scientists tried to add a "gas pedal" using a single antibody that targeted a receptor called CD28. However, this was dangerous. If you press the gas pedal without holding the steering wheel (the cancer target), the car might crash into innocent bystanders (healthy tissues), causing a massive, life-threatening reaction. This happened in a famous medical disaster in 2006.

The Innovation:
The team created a new, super-smart tool called a BiCo (Bivalent Costimulator).

• The Analogy: Think of the BiCo as a safety harness with a turbo boost.
  • It has two "hands." One hand holds onto the cancer cell (just like the TCE does).
  • The other hand holds onto the T-cell's CD28 receptor (the gas pedal).
  • The Magic Trick: The BiCo is designed so that it only presses the gas pedal if it is also holding onto the cancer cell. If there is no cancer nearby, the BiCo stays dormant and doesn't press the gas. This makes it incredibly safe.

What They Found

The researchers tested this on patients with prostate cancer and in mouse models. Here is what happened:

1. The "Wake Up" Call: When they gave patients the TCE alone, the T-cells went to sleep (anergy). But when they added the BiCo, the T-cells woke up instantly. They started multiplying and killing cancer cells again.
2. The Molecular Proof: They looked at the T-cells under a microscope (using a technique called single-cell RNA sequencing). They saw that the "sleeping" T-cells had turned on "stop" genes (like BACH2 and KLF2) and turned off "go" genes. The BiCo flipped the switch back: it turned off the "stop" genes and turned on the "go" genes.
3. Better than the Competition: They compared their new "safety harness" (BiCo) to older, simpler versions (univalent antibodies). The new version was much stronger and more effective, like a V8 engine compared to a bicycle.
4. The Result: In mice with established tumors, the TCE alone couldn't clear the cancer. But the combination of TCE + BiCo wiped out the tumors completely.

Why This Matters

This paper changes how we think about cancer immunotherapy.

• Old Way: We thought T-cells failed because they were "exhausted" (like a marathon runner who ran too far).
• New Way: The paper shows they were actually "paralyzed" (like a runner who was told to stop running). They weren't tired; they were just missing the second signal to keep going.

The Takeaway:
By adding this new "BiCo" tool, doctors can potentially take a treatment that stops working after a few weeks and make it work again. It turns a "sleeping" immune system back into a "fighting" one, potentially making these cancer drugs work for more people and for longer periods.

The team is planning to start human clinical trials in 2026 to see if this "two-signal" strategy works in the real world, promising a future where cancer treatments are both safer and much more effective.

Technical Summary

1. Problem Statement

Bispecific T-cell engagers (TCEs) that target CD3 and tumor-associated antigens (TAAs) are a cornerstone of modern cancer immunotherapy. However, clinical observations and preclinical data suggest that single-agent TCE therapy often leads to T-cell hyporesponsiveness.

• The Phenomenon: Prolonged TCE treatment induces a state of functional paralysis in T-cells characterized by abolished proliferative and lytic capacity.
• The Mechanism: This hyporesponsiveness is distinct from terminal exhaustion (senescence). Instead, it resembles anergy or quiescence, driven by "Signal 1" (TCR/CD3) stimulation without adequate "Signal 2" (costimulation).
• Clinical Consequence: This leads to transient tumor responses followed by relapse, as T-cells fail to sustain antitumor immunity. Current strategies using univalent CD28 costimulators or checkpoint inhibitors have limitations in reversing this specific state or carry safety risks (e.g., cytokine release syndrome).

2. Methodology

The study employed a multi-faceted approach combining clinical sample analysis, single-cell genomics, protein engineering, and preclinical models.

• Clinical Cohort: Peripheral Blood Mononuclear Cells (PBMCs) were collected from five patients with metastatic castration-resistant prostate cancer (mCRPC) treated with a PSMA-directed TCE (CC-1) in a Phase 1a trial (NCT04104607). Samples were taken before treatment and after 7 days of continuous dosing.
• Single-Cell RNA Sequencing (scRNA-seq): Paired samples (Day 1 vs. Day 8) underwent 10x Genomics scRNA-seq to profile transcriptional changes in CD4+ and CD8+ T-cells, focusing on activation, anergy, and exhaustion markers.
• Protein Engineering (BiCo Development):
  • The authors engineered Bivalent Bispecific Costimulators (BiCos). These are tetravalent antibodies comprising:
    • A TAA-binding arm (targeting Endoglin or B7-H3).
    • A bivalent CD28-binding arm (two scFv domains).
  • Rationale: Bivalency is required for potent CD28 agonism, but monovalency is usually required for safety (to prevent off-target activation). The authors designed a format where CD28 bivalency is sterically constrained, allowing agonism only when the antibody is immobilized on a target cell (providing "Signal 1" via a concurrent TCE).
• Comparative Controls:
  • KiH (Knob-into-Hole) Constructs: Univalent CD28 bispecifics were generated to benchmark against the bivalent BiCos.
  • TCE Alone: High and low doses of TCE (CC-1) and the approved DLL3xCD3 TCE (tarlatamab) were used as controls.
• In Vitro & In Vivo Models:
  • In Vitro: PBMCs from patients and healthy donors were co-cultured with tumor cells (LNCaP-E, SHP-77) to assess proliferation, cytokine secretion (IFN-γ, IL-2), and cytotoxicity.
  • In Vivo: Humanized NSG mice xenografted with LNCaP-E tumors were treated with combinations of TCE + BiCos vs. TCE alone. Pharmacokinetics and toxicity were assessed in C57BL/6 mice.

3. Key Contributions

• Identification of TCE-Induced Anergy: The study provides the first clinical evidence that single-agent TCE therapy induces a specific transcriptional signature of anergy/quiescence (upregulation of BACH2, CBLB, KLF2; downregulation of AP-1 factors) rather than terminal exhaustion.
• Novel BiCo Format: Development of a bivalent CD28 bispecific antibody that maintains strict tumor-restricted activity. Unlike previous attempts that risked systemic toxicity, this format requires simultaneous binding to the tumor (via TAA) and the T-cell (via CD3/TCE) to trigger the bivalent CD28 engagement necessary for signaling.
• Reversal of Hyporesponsiveness: Demonstration that adding BiCos to TCE treatment not only prevents but reverses the anergic state in patient T-cells, restoring proliferative and cytotoxic functions.

4. Key Results

A. Clinical Observation of Hyporesponsiveness

• Patients treated with CC-1 showed a rapid but transient drop in PSA, followed by a loss of T-cell responsiveness.
• Functional Loss: Post-treatment T-cells showed reduced proliferation, cytokine secretion, and cytolytic activity upon re-stimulation.
• Transcriptional Signature: scRNA-seq revealed a coordinated upregulation of anergy/quiescence genes (BACH2, CBLB, FOXP1, PTPN22, TXNIP) and suppression of immediate-early activation genes (FOS, JUN, CD69).
• Phenotype Shift: CD8+ T-cells shifted from effector-memory to dysfunctional terminal effector-like states; CD4+ T-cells shifted from central memory to a quiescent/resting state.

B. BiCo Engineering and Specificity

• Four bivalent formats were tested. Only the bs-CD28-4 format (scFv fused to light/heavy chains) displayed strictly target-restricted activity, showing no T-cell stimulation in the absence of TAA binding.
• Two lead candidates were selected: BiCo-1 (EndoglinxCD28) and BiCo-2 (B7-H3xCD28).

C. Superior Efficacy of Bivalent BiCos

• Potency: BiCos combined with low-dose TCE induced significantly higher T-cell proliferation and tumor killing than high-dose TCE alone.
• Comparison to KiH: Bivalent BiCos outperformed univalent Knob-into-Hole (KiH) CD28 constructs. This was attributed to a 5-fold slower off-rate of the bivalent CD28 binding, leading to more stable complex formation and sustained signaling.
• In Vivo Efficacy: In humanized mice with established tumors, the combination of TCE + BiCo eliminated tumors and significantly improved survival, whereas TCE alone failed to control tumor growth.

D. Reversal of Anergy in Patient Samples

• Restoration of Function: When BiCos were added to PBMCs from TCE-treated patients (who were hyporesponsive), T-cell proliferation and killing were restored to levels exceeding those of responsive cells treated with TCE alone.
• Transcriptional Reversal: BiCo treatment reversed the anergic signature:
  • Downregulated: BACH2, KLF2, PNRC1, TXNIP.
  • Upregulated: Cell-cycle genes (MCM family, CDC genes) and effector genes (GZMB, NKG7, IFNG).
• Generalizability: The effect was replicated with tarlatamab (DLL3xCD3), suggesting this is a class-wide effect of TCEs that can be mitigated by CD28 costimulation.

5. Significance

• Mechanistic Insight: The study redefines the mechanism of TCE failure in solid tumors, identifying anergy/quiescence (driven by lack of Signal 2) as a primary barrier, distinct from exhaustion.
• Therapeutic Strategy: It establishes conditional CD28 costimulation as a viable strategy to overcome TCE-induced hyporesponsiveness. By delivering "Signal 1" (TCE) and "Signal 2" (BiCo) simultaneously and restrictively to the tumor site, the therapy maximizes efficacy while minimizing systemic toxicity (CRS).
• Clinical Impact: The findings support the initiation of clinical trials (planned for 2026) combining TCEs with BiCos targeting different TAAs. This approach aims to convert transient responses into durable antitumor immunity, potentially expanding the utility of TCEs to a broader range of solid tumors.
• Safety: The bivalent BiCo format successfully overcomes the historical safety hurdle of CD28 superagonists (TGN1412 incident) by ensuring agonism only occurs in the context of tumor targeting.