T-Cell Redirecting Tri-Specific Antibodies for Solid Tumor Treatment Notes

T-Cell Redirecting Tri-Specific Antibodies for Solid Tumor Treatment

Objective

• Determine whether Tri-specific antibodies (tsAb) that simultaneously block immune checkpoint proteins (PD-L1 and LAG-3?) while redirecting CD3+ T Cells work better in enhancing T cell activation and solid tumor cell death than current bispecific antibodies (bsAb).

Background

• Immunotherapy is a relatively new form of cancer treatment.
• T-Cell Redirecting Bispecific Antibody (bsAb) is a type of immunotherapy.
  • Has been proven to work in blood cancers like leukemia.
  • Less effective in solid tumors.
• Challenges with solid tumors:
  • Tumor immunosuppressive microenvironment.
  • Immune checkpoint proteins that help tumors evade the immune system.
• T-Cell Redirecting Tri-Specific antibody (tsAb) has three targets.
  • Current tsAbs mostly target two tumor-associated antigens and one antigen on the T cell.
  • Proposed strategy: target an immune checkpoint protein on the cancer cell, block it, and then target a T-Cell.
  • This could help suppress the immunosuppressive tumor microenvironment.

Review of Literature

• Article 1: A novel CD19/CD22/CD3 tri-specific antibody… (liquid tumor).
• Article 2: A novel engineered EpCAM/CD3/4-1BB tri-specific antibody… (solid tumor).
• Article 3: The redirected killing of PDL1…(bispecific).

Research Question

• Does a tri-specific antibody that simultaneously blocks immune checkpoint proteins while redirecting CD3+ T cells work better in enhancing T cell activation and tumor cell death than current bispecific antibody treatments?

Methods

• Solid tumor cell line.
• Treatment groups (3 each):
  1. Control (no treatment).
  2. bsAb alone (EpCAM and CD8).
  3. Anti-PDL-1 antibody alone.
  4. Combination tsAb (bsAb + anti-PD-L1).
• T cells: Primary CD3+ T cells isolated from healthy donors.
• Measurements:
  • Tumor cell capability.
  • T cell activation.
  • Cytokine levels.
  • Apoptosis in tumor cells.

Expected Results

• Expected results:
  • Combination therapy resulted in the greatest reduction in tumor viability, highest T cell activation, and elevated cytokine production.
  • tsAb worked the best out of everything.

Conclusion

• tsAbs that both bind to and block the checkpoint inhibitors on cancer cells enhance the immune response and overcome immunosuppression in solid tumors.
• This strategy could improve the effectiveness of T Cell redirecting therapies.

JNJ Article: Creating the Antibody (JNJ-78306358)

• Scientists engineered a bispecific antibody that can bind to:
  • CD3 on T cells
  • HLA-G on tumor cells.
• It acts like a bridge bringing T cells close to tumor cells so they can kill them.

How They Measured Everything

Where is HLA-G Found?

• Technique: Immunohistochemistry (IHC).
  • Purpose: Check which human tissues have the HLA-G protein.
  • How it works:
    • They used a dye-labeled antibody (called 4H84) that sticks to HLA-G.
    • They applied it to thin slices of tissue from organs and tumors.
    • Under a microscope, brown staining = HLA-G present.
    • This helped them learn that HLA-G is mostly in tumors, placenta, and pituitary glands, but not much in other healthy tissues.

How Strongly Does the Antibody Bind?

• Techniques: Flow Cytometry & HDX-MS (Mass Spectrometry).

Flow Cytometry

• Purpose: Measure how well the antibody binds to cells and if it causes T cells to get activated or kill.
• How it works:
  • Cells are mixed with the antibody.
  • Cells go one-by-one through a laser beam.
  • If the antibody sticks to a cell, it glows due to a special fluorescent tag.
  • They can measure:
    • How much HLA-G a cell has.
    • Whether T cells get activated (CD25 turns on when T cells are activated).
    • Whether cancer cells die after treatment.

HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry)

• Purpose: Find out exactly where the antibody binds on HLA-G.
• How it works:
  • Parts of HLA-G that are covered by the antibody change more slowly in heavy water (D_2O).
  • Mass spectrometry detects those changes, revealing the "landing spot" of the antibody.

Can It Block Cancer's Defense System?

• Technique: Receptor Blocking Assay
  • Purpose: See if JNJ-78306358 can block HLA-G from stopping the immune system.
  • How it works:
    • They used cells with HLA-G receptors (called ILT2 and ILT4).
    • Added HLA-G and saw if it would bind.
    • Then added JNJ-78306358 to see if it blocks that binding (it did!).

Does It Kill Tumor Cells?

• Technique: In Vitro Killing Assay (lab dish)
  • Purpose: Check if T cells will destroy cancer cells when JNJ-78306358 is added.
  • How it works:
    • Mix cancer cells with T cells and the antibody.
    • After a few days, measure how many cancer cells died.
    • More cancer cell death = better performance.
    • They used different doses and different cancer cell types.

Does It Work in Living Animals?

• Technique: Mouse Studies (in vivo)
  • Purpose: See if JNJ-78306358 kills tumors in live animals.
  • How it works:
    • Mice were given human tumors and human T cells.
    • They injected the antibody and measured:
      • Tumor size over time (with rulers or imaging).
      • How many T cells entered the tumor (using IHC and flow cytometry).
      • Antibody levels in the mouse’s blood and tumors (to check how long it lasts = pharmacokinetics).

How Much HLA-G is Needed to Work?

• Techniques:
  • Wes (a small version of Western blot) to measure protein levels.
  • Cytokine tests (like IFN-γ release) to see how strong the T cell response is.
  • Result: They found that the therapy only works well if the tumor has enough HLA-G on its surface.

Summary of Tools They Used

Article 1: A novel CD19/CD22/CD3 trispecific antibody enhances therapeutic efficacy and overcomes immune escape against B-ALL

• CD19/CD3 bispecific antibody: blinatumomab shows success in leukemia
• Really bad relapse though because of the loss of the CD19 TAA
• Researchers developed a CD19/CD22/CD3 Tri specific antibody to address this
• Methods:
  • Fused anti-CD19 & anti-CD22 --> anti-CD3
  • They made many formats of bsAbs and tsAbs to find the best configuration
  • CC format was best: CD19/FMC63 to C terminus of CD3 heavy chain; CD22/Nb25 to C terminus of CD3 light chain
• In Vitro Testing:
  • Nalm6 cell line (B-ALL cell line with CD19 and CD22 expression)
  • Assays
    • Cytotoxicity (how well antibody caused T cells to kill cancer cells)
    • Cytokine production: indicate T cell activation
    • Synapse formation: see if tight contacts form between T cells and cancer cells
    • Binding: flow cytometry to make sure that each antibody binds correctly
• In Vivo Testing:
  • NCG mice lacking immune cells
  • Mice injected with B-ALL cells initially; few days later human T cells
  • Treated with either tsAb, bsAb (CD19/CD3 or CD22/CD3), or combination
  • Measurements:
    • Tumor burden: bioluminescent imaging (B-ALL cells have luciferase enzyme)
    • Survival of mice
    • Cytokines in blood
    • How long T cells lasted in the mice
  • Immune escape model: mixed tumor with CD19 and CD22 to simulate antigen loss
    • Only tsAb could target both AND prevent tumor relapse

More In Vivo

• B-ALL cells from a real patient were injected into the mice
• Medium CD19 and low CD22.
• tsAb worked better with this even when bsAbs were used together.
• Mice with tsAb had longer survival AND less relapse
• Compared tsAb with blinatumomab: both killed CD19+ cells well
• Only tsAb could kill CD19-/CD22+ cells
• This tsAb could have lots of potential
• Researchers created a tsAb targeting CD19, CD22 TAAs and CD3+ T cells to overcome antigen loss in solid tumors
• In vitro tests: tsAb effectively activated T cells, promoted cytotoxicity, and formed strong immune synapses
• In vivo tests: tsAb reduced tumor burden in immunodeficient mice, prolonged survival, and prevented relapse better than tested bsAbs
• tsAb only therapy that worked in models where either CD19 or CD22 were missing
• Compared to current bsAb, blinatumomab, tsAb eliminated wider variety of cells Ex: CD19-/CD20+
• This tsAb shows strong promise in leukemia therapy with reduced relapse risk

Article 2: The redirected killing of PD-L1 positive tumor cells by expanded MAIT cells is mediated with a bispecific antibody targeting TCR Vα7.2 and PD-L1

• Currently, most bsAbs target CD3 (also on regulatory T cells that suppress immune responses)
• Looked into targeting MAIT cells (rare immune cells; type of T cell)
• Methods:
  • White blood cells from healthy donors
  • Stimulated the cells with antibodies specific to MAIT cells
  • Also with cytokines to help the MAIT cells grow
  • 90% of cells were now MAIT cells
  • Most had CD8, naïve phenotype, and low inhibitory markers
  • bsAb: one arm to Vα7.2 on MAIT and other to PD-L1 on tumor
  • PD-L1+ tumor cell lines (lung/ovarian cancer)
  • Engineered to express luciferase so it could be measured by light
  • Binding tests
    • Tumor binding: very strong binding
    • MAIT binding: okay
    • MAIT x Tumor bridging: worked!
  • Tested if the bsAb could turn on the MAIT cells only in presence of tumor (yes)
  • Tested tumor cell killing (less light = more killing)
  • bsAb had strong tumor killing in the PD-L1 cell lines

Main Ideas:

• This study explored targeting MAIT cells, a rare type of T cell, instead of CD3
• Created a novel bsAb binding to Vα7.2 on MAIT cells and PD-L1 on tumor cells
• PD-L1 (programmed death-ligand 1) is often overexpressed on cancer cells to inhibit T cell response
• Engineered tumor cell lines (lung and ovarian) to express PD-L1
• This bsAb only activated the MAIT cells in the presence of the PD-L1+ tumor cells
• This bsAb showed strong tumor killing in these cell lines

Article 3: IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer

• Colorectal cancer is one of the most common digestive system cancers
• EpCAM is very much expressed in over 97% of CRC tumors
• Needed for cell adhesion, signaling, proliferation, differentiation
• Designed tsAb targeting EpCAM/CD3/4-1BB
• 4-1BB co-stimulatory receptor on activated T cells, NK cells, other immune cells
• Boosts T cell proliferation (especially in CD8+), enhances cytokine production, prevents T cell exhaustion/death, promotes formation of memory T cells
• Wanted to activate T cells only in presence of EpCAM
• Measured:
  • Tumor cell killing
  • Cytokines
  • Effector memory T cells
  • Anti-apoptotic protein expression
• Mice transplanted with human blood cells and tumors; tsAb injected daily for 11 days
• Tracked with bioluminescent imaging, tumor volume, and survival of mice
• Target specific binding:
  • EpCAM: high affinity
  • 4-1BB: high affinity
  • CD3: low affinity (on purpose)
• Tumor specific T cell activation confirmed!
• CD8^+ T cells are essential
• CD8^+ T cells are the main killer
• Main cells that this tsAb activates to attack the cancer isCD8^+

Main Ideas:

• Novel trispecific antibody
• Over 97% of colorectal cancer (CRC) tumors express EpCAM
• Researchers developed a tsAb targeting EpCAM, CD3, and 4-1BB
• 4-1BB is a co-stimulatory receptor on activated T cells and NK cells that boosts CD8^+ T cells
• Goal: activate T cells only in presence of EpCAM on tumor cells
• Results showed tumor-specific T cell activation
• However, activating CD3 on the T cell as well as a T cell booster can lead to overactive immune response
• EpCAM/PD-L1/CD3