Bispecific antibodies: on-target off-tumor Toxicity and Overcoming strategies
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Bispecific antibodies: on-target off-tumor Toxicity and Overcoming strategies
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Bispecific antibodies: on-target off-tumor Toxicity and Overcoming strategies.
CD3XTAAs bispecific antibodies, among which tumor-associated antigens (TAAs) are mostly tumor cell membrane proteins, which need to be highly expressed in tumors and not expressed in healthy cells, otherwise they are prone to on-target off-tumor toxicity.
Tumor antigen
Membrane antigen
But the reality is that most membrane proteins are conserved between tumor cells and healthy cells. The ideal TAAs are tumor neonatal antigens or tumor virus antigens, but they are limited to a small part of tumors.
Nowadays, the most commonly used target is that tumor cells overexpress antigens (such as CEA, EGFR, EpCAM and HER2), and healthy cells have low expression, so there will still be varying degrees of off tumor toxicity.
Intracellular antigen
Some of the most different expressions in cancer are intracellular proteins, but traditional antibodies cannot enter the cell.
Such intracellular proteins need to be presented by HLA class I molecules to be recognized. These surface peptide/HLA complexes can be targeted with peptide-specific antibodies or TCR molecules, such as ImmTACs or TCR-like antibodies.
The TCR arms of ImmTACs or TCR-like antibodies have enhanced affinity to a low nM level to better recognize TAAs and facilitate the CD3 arm to better recruit T cells to kill tumor cells.
However, the main disadvantage of TCR-like CD3-double antibodies is that HLA is restricted and tumors usually down-regulate HLA. In addition, on-target off-tumor side effects can still be found.
For example, clinical trials of tebentafusp have found dose-limiting toxicity cases.
In order to improve tumor selectivity and specificity, and reduce off tumor toxicity, many researchers began to optimize the affinity of CD3 antibodies and TAAs.
For example, the effectiveness of TAA can be improved by producing so-called 2:1CD3-BsAbs. These 2:1CD3-BsAb antibodies contain a second TAA binding fragment to enhance the binding to tumor cells, but when the TAA affinity is too high, it can Combines with low-level TAA expressed on healthy cells.
On the other hand, if the TAA affinity is too low, the efficacy of CD3-BsAb will be compromised. Therefore, adjusting TAA affinity achieves a balance between specificity and effectiveness.
Common strategies
Prodrug technology
Utilizing the characteristics of tumor microenvironment (such as low pH, specific ECM components, protease, etc.) to develop prodrugs is an important strategy in overcoming off-tumor toxicity in recent years.
1. Probody technology
The Probody technology of CytomX Therapeutics adds a masking peptide to the N-terminus of the antibody through an enzymatic linker.
When the antibody reaches the tumor microenvironment, it can be enzymatically removed by the tumor-specific protease to remove the masking peptide, thereby binding to the target. In the study of cynomolgus monkeys, the maximum tolerated dose of Probody antibody was 60 times that of unmasked peptides.
Probody working principle
2. COBRA technology
Maverick Therapeutics’ COBRATM double antibody consists of two half antibodies:
A half-antibody consists of an active anti-CD3 single-chain variable region (scFv VH), which is linked to an inactive variable region fragment (VLi) at the C-terminus through a linker that can be digested by MMP9, and a tumor-associated antigen antibody is connected to the N-terminus .
A half-antibody consists of an active anti-CD3 single-chain variable region (scFv VL), which is linked to an inactive variable region fragment (VHi) at the C-terminus through a linker that can be digested by MMP9, and a tumor-associated antigen antibody is connected to the N-terminus .
COBRA working principle (document 2)
When the tumor reaches the local area, the half-antibody is enzymatically hydrolyzed between VH and VLi, VL and VHi by MMP9 in the tumor microenvironment. The active fragment of CD3 antibody is exposed, and the tumor is recognized by the tumor-associated antigen antibody (aTTA). The active fragment of CD3 antibody recruits and activates T cells to exert anti-tumor activity.
3. Half antibody + half antibody
It is similar to the COBRA technology of Maverick Therapeutics. It is also two half antibodies. One end of the anti-CD3VH or anti-CD3VL antibody is connected to the target antigen antibody, but the other end is not added with other masking sequences (such as inactive CD3 or masking peptides, etc.).
The light and heavy chains in the variable region of CD3 in the body combine and assemble to form a double antibody. This double antibody carries two different target antigen single-chain variable region fragments (scFv1 and scFv2).
4. Roche’s pro-FOLR1-TCB
FOLR1-TCB is based on Roche’s 2+1 CD3XFOLR1 double antibody. The anti-CD3 antibody is connected to anti-idiotypic anti-CD3 through a protease cleavable linker to form the pro-FOLR1-TCB.
Pro-FOLR1-TCB is cleaved by the tumor protease linker in the tumor microenvironment to release the active form of FOLR1-TCB.
pro-FOLR1-TCB
Change the affinity of CD3 antibodies
For the CD3XHER2 double antibody, the in vivo distribution study using SPECT showed that the double antibody with high affinity CD3 is mainly distributed in the T cell enrichment area (spleen and lymph nodes), and the low affinity CD3 double antibody is more distributed in the tumor.
CD3 affinity and the distribution of double antibodies
Intratumoral injection of CD3 bispecific antibody
Although it is not possible to completely organize the spread of CD3 dual antibody to surrounding tissues, intratumoral injection is an effective way to reduce on target off tumor. Although it is possible to inject drugs into local non-superficial tumors under ultrasound guidance, this method is still complicated and requires multiple injections.
Other methods
In addition, some research units have developed cells that secrete bispecific antibodies (STAb-T), which only secrete bispecific antibodies after entering the tumor microenvironment and being activated.
The use of oncolytic viruses or nanoparticles to encapsulate drugs and release them locally to the tumor is also the direction of development.
However, these methods are still in the early stage of research.
Expert Comments:
On target off-tumor toxicity limits the use of antibodies. With the in-depth study of the tumor microenvironment, some prodrug technologies began to emerge, Roche also participated, Takeda acquired Maverick Therapeutic, and obtained COBRATM double antibody technology.
(source:internet, reference only)
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