June 23, 2021

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CD28 double antibody + CD3 double antibody = CAR-T?

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CD28 double antibody + CD3 double antibody = CAR-T?

CD28 double antibody + CD3 double antibody = CAR-T?   The success of the Blinatumomab (CD19xCD3) bispecific antibody led to the development of bispecific antibodies. At present, more than half of the bispecific antibodies in development are activated by CD3 to activate T cells, and use T cells to kill target cells to treat diseases. the goal of.

Although this type of bispecific antibody occupies an important position in immunotherapy, some cross-over studies have shown that compared with CAR-T cell therapy, CD3 bridged bispecific antibody still has a certain gap in efficacy.

One of the reasons for this is that the CD3 type bispecific antibody activates T cells mainly through “signal 1”, while the activation of T cells by CAR-T includes “signal 1” and “signal 2.”

“Signal 2” plays a huge role in T cells releasing cytokines, T cell survival and differentiation.

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 1 Immune synapse activation of T cells mediated by signal 1 and signal 2

Compared with CAR-T cell therapy, bispecific antibodies have the advantages of being well tolerated (relatively low CRS) and versatility. Therefore, how to modify bispecific antibodies to improve their curative effect without compromising the existing advantages is the dual A direction of specific antibody research.

The author of this article enhances the efficacy of CD3 bispecific antibodies by introducing a second bispecific antibody that can bind to CD28. The CD3 bispecific antibody mainly activates T cells through “signal 1”, while the CD28 bispecific antibody mainly uses “signal 1”. Signal 2″ enhances the therapeutic effect of T cells.

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 2 Schematic diagram of the mechanism of TSAxCD3 and TSAxCD28 bispecific antibodies to activate T cells

1. Immune Synapse Mediated by TSAxCD3 and TSAxCD28 Bispecific Antibody

TSAxCD28 bispecific antibody bispecific antibody requires TSAxCD28 and TSAxCD3 bispecific antibody to bind to target cells and T cells at the same time to form an immune synapse.

In order to test whether these two bispecific antibodies can form immune synapses between target cells and T cells and play a role, the authors constructed a 293 cell line expressing human CD20/PSMA as target cells, and constructed CD20xCD3 and PSMAxCD28 bispecifics. Antibody.

Immunofluorescence experiments have shown that when only target cells or T cells are present, bispecific antibodies can bind to cells and be evenly distributed on the cell surface (Figures 3A and 3B). When T cells and target cells are present at the same time, the two bispecific antibodies Antibodies can mediate the formation of immune synapses (Figure 3C).

Compared with medication alone, the combined use of two bispecific antibodies can strongly promote the proliferation of T cells (Figure 3D and 3E).

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 3 Immune synapse mediated by double antibodies

2. TSAxCD28 bispecific antibody increases the killing of tumor cells by TSAxCD3 bispecific antibody

The killing effect of bispecific antibodies with different targets (PSMA and MUC16) on different tumor cells (prostate cancer cell C4-2 cells express PSMA antigen; ovarian cancer cell PEO-1 express MUC16) proved that when the TSAxCD28 bispecific antibody and When TSAxCD3 antibody is used in combination, the killing effect of TSAxCD3 on tumor cells is greatly improved (Figure 4B and 4G)-PSAMxCD28 makes the EC50 of PSMAxCD3 about 50 times lower; MUC16xCD28 increases the maximum killing effect of MUC16xCD3 from 40% to 80 %.

In addition to enhancing the killing of tumor cells by CD3-related double antibodies, TSAxCD28 also improves the release of IFN-γ and the activity and proliferation of CD4 and CD8 T cells (Figure 4C-4E and 4H-4J).

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 4 TSAxCD28 increases the anti-tumor activity of TSAxCD3 bispecific antibody

3. TSAxCD28 enhances the antitumor activity of TSAxCD3 bispecific antibody in vivo

In immunodeficiency mouse tumor models, although MUC16xCD3 alone can inhibit the growth of tumors to a large extent, it cannot completely eliminate tumors, but the combination of MUC16xCD28 and MUC16xCD3 can basically completely eliminate tumors. Is lasting.

However, EGFRvIIIxCD3 and EGFRvIIIxCD3+ MUC16xCD28 as controls were completely unable to grow a tumor (Figures 5A and 5B). After treatment, mice in the MUC16xCD28 and MUC16xCD3 combination group also had significantly lower tumor antigen load than other antibody treatment groups (Figure 5C).

Similarly, in a humanized mouse model expressing human CD3/CD28 and related tumor antigens, the combination of TSAxCD28 bispecific antibody and TSAxCD3 antibody has stronger anti-tumor activity than the two alone.

Moreover, the combination can promote T cells to release more IFN-γ and IL-2 (Figure 6A-6F).

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 5 MUC16xCD28 enhances the anti-tumor activity of the MUC16xCD3 bispecific antibody in the NGS model tumor model

CD28 double antibody + CD3 double antibody = CAR-T?
Figure 6 TSAxCD28 double antibody enhances the anti-tumor activity of TSAxCD3 double specific antibody in a humanized mouse model

4. Safety of TSAxCD28 bispecific antibody

The dry and wet fixation test methods recommended by the FDA were used to detect the activation of T cells by CD28-related antibodies. The bispecific antibodies (PSMAxCD28, MUC16xCD28 and their parent antibodies) used in this study were not able to promote the proliferation of T cells, and were used as controls. The positive antibodies CD28-SA (bivalent CD28 super mobile monoclonal antibody) and CD3 monoclonal antibody can greatly promote the proliferation of T cells.

In monkey experiments, the TSAxCD28 bispecific antibody alone could not promote the release of IFN-γ, IL-2, IL-6, IL-8 and IL-10 and other cytokines, while CD28-SA can promote the release of related cytokines ( Figure 7C-7G), which proves that the use of TSAxCD28 alone is safe and controllable.

Figure 7 Activation of T cells by TSAxCD28 bispecific antibody

Sumu up: 

CD3-related bispecific antibodies have achieved great success in the treatment of hematological tumors, but their therapeutic effects in the field of solid tumors are not satisfactory. Therefore, there are many areas that need to be improved, such as enhancing its tumor killing effect and reducing CRS and other related side effects.

In terms of enhancing anti-T cell activity, people have long realized that T cells activated by the TCR complex (signal 1) can enhance the ability to eliminate tumor cells under the action of costimulatory molecules (such as CD28, etc.).

However, the early technical backwardness and the TGN1412 clinical accident in 2006 led to the termination of CD28-related agonistic antibody research.

With the advancement of antibody technology and the further deepening of understanding of CRS and other related side effects, CD28-related antibodies have gradually entered people’s field of vision.

Regeneron constructed TASxCD28 bispecific antibodies. These antibodies are different from the early CD28 super agonistic antibodies. They can not only mediate the formation of signal 2 related immune synapses, but also will not systemically activate T cells when used alone. Therefore, it is safer.

When combined with the TSAxCD3 bispecific antibody, it can greatly improve the efficacy of the CD3-related bispecific antibody, and can increase memory T cells and activated T cells in tumors.

The combination of TSAxCD28 bispecific antibody and TSAxCD3 antibody can not only activate signal 1 through CD3 but also activate signal 2 through CD28, which is functionally similar to the effect of CAT-T cell therapy (CAR-T activates T cells through CD3ζ in the package, And through the CD28 and other related costimulatory molecules in the package to promote the proliferation and effect of T cells).

However, compared with CAR-T, the combination of bispecific antibodies has more advantages. First, the antibody preparation is easier and universal. Second, when the tumor antigens recognized by the two antibodies are different (TSA1xCD28, TSA2xCD3), there is only one target. There are two antigens on the cells to maximize the activation of T cells, thereby reducing the off-target effect of the double antibody.

This research gave us a new understanding of the role of CD28-related antibodies. Perhaps in the near future, CD28 antibodies will come back to life!

(source:internet, reference only)

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