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Bispecific antibodies for the treatment of triple-negative breast cancer

Bispecific antibodies for the treatment of triple-negative breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking expression of estrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 (HER2), and has increased metastatic potential Related to poor prognosis.

Although systemic chemotherapy, radiotherapy and surgical resection are still the current treatments for TNBC patients, the immunogenicity of this aggressive disease provides opportunities for the development of TNBC targeted immunotherapy.

The treatment of TNBC based on bispecific antibodies has recently attracted attention in the scientific community. The FDA-approved bispecific T cell junction agent blinatumomab has previously established a clinical precedent for acute lymphoblastic leukemia.

Unmet medical needs for triple-negative breast cancer treatment

TNBCs account for about 15% of all breast cancers. Although the improvement of patient survival rate is limited, population-targeted therapy for TNBC patients has become the main treatment modality (Box 1).

Recent studies have shown that the regulation of the immune system may provide a better way to treat TNBC, because the progress of TNBC is due to its complex interaction with the immune system. The existence of cytotoxic tumors in filtered lymphocytes in TNBC tumors is recognized and is associated with a good prognosis of immunotherapy.

Other attributes predicting the response of TNBC patients to immunotherapy include high mutation burden, defects in mismatch repair mechanisms, microsatellite instability, and expression of immune checkpoint molecules.

The concept of using the human immune system to treat TNBC is developing and growing. This can be demonstrated by exciting research such as adoptive cell therapy, immune checkpoint inhibitors, oncolytic viruses, cytokines, antibody-drug conjugates, and cancer vaccines. . Recent research has focused on the development of TNBC multifunctional bispecific antibodies. (I) Redirect the immune cell population to TNBC cells, or (ii) bind to key receptors on TNBC cells at the same time (Table 1, Key Table).

Bispecific antibodies for cancer immunotherapy

About 86% of bispecific antibody therapies in the clinical pipeline are used for tumor treatment. Advances in recombinant DNA technology have produced a wide variety of bispecific antibodies against cancer (Figure 1). The vast majority of bispecific antibodies currently used clinically for cancer treatment have a dual-participation mechanism of immune cells and tumor cells, and are usually formatted as BiTEs.

The FDA approved blinatumomab, the first class of drugs for the treatment of B-cell malignancies, setting a clinical precedent for the development of bispecific antibodies in the oncology field. Structurally, BiTEs are designed to contain immunoglobulin-derived single-chain variable fragments (scFv), which contain the variable heavy chain (VH) and variable heavy chain (VH) of an antibody against tumor-associated antigen (TAA).

Light chain (VL) region, the antibody is linked to the scFv of the antibody targeting T cells. Blinatumomab is an anti-CD3/anti-CD19 antibody that simultaneously targets low-affinity CD3-expressing T lymphocytes and high-affinity CD19-expressing leukemia cells, so that these two cell types are very close, thereby reducing the cytotoxicity of T cells Redirect to tumor cells (Figure 2).

A key advantage of BiTE-based antibody therapy is that the cytolytic activity of T cells can be redirected to tumor cells without relying on T cell receptor (TCR) specificity, costimulatory signals, or peptide antigen presentation. Compared with B-ALL patients receiving chemotherapy, the overall survival (OS) of patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) treated with blinatumomab was 7.7 months, and the OS of the latter was reduced by 4 Months.

The safety and safety of blinatumomab combined with immune checkpoint inhibitors (including anti-cytotoxic T lymphocyte-associated protein-4 (CTLA-4) monoclonal antibody ipilimumab and anti-programmed death 1 (PD-1) monoclonal antibodies nivolumab and pembrolizumab) Effectiveness is being evaluated in clinical trials (ClinicalTrials.gov identification numbers: NCT02879695, NCT03340766, NCT03512405, NCT03605589).

Some bispecific antibodies are designed to activate CD3+ T lymphocytes and target them to HER2-expressing tumors. The new asymmetric anti-CD3/anti-HER2 bispecific antibody M802 recently demonstrated targeted cytotoxic activity against HER2-positive cancer cells, accompanied by cytokine secretion. In addition, a heterogeneous gastric cancer mouse model was used to observe tumor growth inhibition in vivo.

The T-cell-dependent bispecific (TDB) antibody BTRC4017A and the BEAT® (bispecific binding of T-cell receptor-based antibodies)-based bispecific antibody ISB1302 are two of the clinical advancements of the CD3×HER2 bispecific. Examples (clinical trials.gov are NCT03983395 and NCT03448042 respectively). A subpopulation of innate pro-inflammatory T-cells, called γδ (γδ) T cells, has recently become an alternative target for immune cell binding to bispecific antibodies.

The bispecific nanobody construct targeting epidermal growth factor receptor (EGFR) and Vγ9Vδ2t cells retains the original keratinocytes representing EGFR while inducing the cytotoxicity of patient-derived colorectal cancer cells. In addition, Fcγ receptors (FcγRs) expressed on natural killer (NK) cells or macrophages are also targeted to tumor cells. The clinical safety and effectiveness of tri-specicNK cell engager therapy DF1001 are being evaluated in the first human multi-part phase I/II trial for the treatment of HER2-positive solid tumors (ClinicalTrials.gov identification number NCT04143711).

In addition to redirecting immune cell populations to tumor cells, bispecific antibodies used in cancer immunotherapy can also trigger other mechanisms of action, such as being a carrier for payload delivery. Radioimmunotherapy and antibody-drug conjugates undergo a mechanism in which a payload containing an isotope or drug, respectively, is directly bound to an antibody against TAA.

After the antibody binds to TAA, the payload is directly delivered to the tumor. In addition, another mechanism of bispecific antibodies is to double block two identical or different receptors expressed on cancer cells. ZW25 is a dual antigen bispecific antibody-drug conjugate targeting two epitopes on HER2. It is currently undergoing a multicenter open-label Phase II trial for the treatment of HER2-expressing gastroesophageal adenocarcinoma (ClinicalTrials.gov Identification number NCT03929666).

Using bispecific antibodies to target multiple co-localized receptors on the surface of cancer cells is a promising method to enhance targeting specificity and minimize targeted toxicity to healthy tissues.

In summary, bispecific antibodies can be achieved by redirecting the cytotoxicity of immune cells to tumor cells, delivering cytotoxic payloads to tumor cells, or simultaneously binding two functionally important receptors on the same cancer cell. A powerful treatment for cancer.

Bispecific antibody therapy for the treatment of TNBC

Immune cell redirection bispecific antibodies in TNBC In preclinical studies of TNBC treatment, most bispecific antibodies are classified as CD3+ T cell adaptors (Figure 3A). Using the dock-and-lock technology platform, a new type of bispecific antibody trophoblast cell surface antigen 2 (Trop2) or CD3×carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5) for CD3 is newly produced.

Treatment of 3D TNBC spheres expressing Trop2 and CEACAM5 with CD3×Trop2 or CD3×CEACAM5 bispecific antibody combined with human peripheral blood mononuclear cells (PBMC) can significantly inhibit the growth of TNBC cells. Interestingly, the addition of an antagonistic anti-PD-1 monoclonal antibody to this model further enhanced cell death in the 3D-TNBC sphere.

These findings provide a proof of concept (POC) that combining T cell redirecting bispecific antibodies and immune checkpoint inhibitors is a feasible way to improve the anti-tumor effect of TNBC and overcome the immunosuppressive tumor microenvironment (TME). Ephrin receptor A10 (EphA10) is a receptor tyrosine kinase, which is overexpressed on 67% of TNBC cells and almost not expressed in normal breast tissues.

The CD3 redirection strategy has recently been used to target EphA10 on TNBC cells with higher efficacy. By fusing single-chain antibody fragment a (VL chain of EphA10 to VH chain of CD3) and single-chain antibody fragment B (VL chain of CD3 connected to VH chain of EphA10), the CD3×EphA10 bispecific antibody is formed into a diabetic body . The addition of CD3×EphA10 bispecific antibody to EphA10-expressing TNBC cells co-cultured with PBMC resulted in T cell-mediated TNBC cell directed lysis.

Another bispecific antibody platform called DART has recently been used as a scaffold to generate bispecific antibodies against CD3 on T lymphocytes and P-cadherin on tumor cells. CD3×P-cadherin molecule PF-06671008 has shown in vivo efficacy in a TNBC mouse model of patient-derived xenograft (PDX) implanted with circulating human T lymphocytes, as evidenced by the regression of TNBC tumors.

In fact, an open phase I dose escalation study is ongoing to evaluate the safety and tolerability of PF-06671008 in TNBC patients expressing P-cadherin (ClinicalTrials.gov identification number NCT02659631).

Epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein detected in more than 90% of breast cancers. It is related to the poor prognosis and treatment resistance of TNBC. Catumaxomab is a multifunctional bispecific antibody that can bind to CD3 on T lymphocytes and EpCAM on tumor cells. It has recently been preclinically studied in TNBC.

Pretreatment with CD3×EpCAM bispecific antibody and subsequent addition of activated T cells resulted in the elimination of drug-resistant EpCAM positive TNBC cells. Methods to enhance the targeting of EGFR to TNBC cells have also been recently evaluated. Blocking immune checkpoint receptor T cell immune receptors with immunoglobulin and ITIM domain (TIGIT) or its ligand poliovirus receptor (PVR) can enhance the cytotoxicity of CD3×EGFR to TNBC cells expressing EGFR active.

The application of exons as antibody therapy carriers is discussed. Retargeting the SMART-Exos nanomedicine platform using synthetic multivalent antibodies to target cytotoxic T cells into TNBC cells. Smart Exos, expressing CD3 and EGFR targeting antibodies In in vitro cytotoxicity tests and human TNBC xenograft mouse models, SMART Exos expressing CD3 and EGFR targeting antibodies can cross-link T cells and EGFR-positive TNBC cells, and induce effective Anti-tumor immune response.

The next-generation SMART Exos platform provides POC for exon-mediated multispecific antibody therapy to treat TNBC.

Although there are some examples of bispecific CD3+ T cell adaptors used to treat TNBC, another type of bispecific antibody that can bind to immune cells to redirect to TNBC tumors has recently emerged. A Fab-like bispecific antibody targeting TNBC cells expressing mesothelin and simultaneously binding to CD16 (FcγRIII) was prepared. CD16 is an activated receptor that is highly expressed on NK cells.

It binds to the Fc region of antibodies that bind to tumor antigens to produce a typical antibody-dependent cellular cytotoxicity (ADCC) mechanism. In fact, the CD16×Mesothelin bispecific antibody mediates the recruitment of NK cells and their filtration into mesothelin-expressing TNBC tumor spheres (Figure 3A). In addition to secreting cytokines, the killing effect of ADCC on mesothelin-positive TNBC cells was also observed.

In addition, the in vitro anti-tumor activity of CD16×Mesothelin was verified by PBMC humanized NOD-SCIDγ (NSG) mouse orthotopic xenograft model, proving that TNBC growth was significantly reduced.

Bispecific antibodies against key receptors expressed on TNBC cells

The use of bispecific antibodies to mediate TNBC cell function at the same time has been shown to be a promising method for the treatment of TNBC (Figure 3B). Recently, a bispecific diabetic Fc fusion protein targeting human epidermal growth factor receptor 3 (HER3), another receptor tyrosine kinase expressed on EGFR and TNBC cells, has been produced. In vitro monolayer cell culture experiments and more complex and physiologically relevant three-dimensional sphere models show that the EGFR×HER3 bispecific antibody can effectively inhibit the proliferation of TNBC cells.

In addition, EGFR×HER3 bispecifically inhibits the survival and expansion of TNBC cancer stem cells (CSCs) in the orthotopic MDA-MB-468 TNBC mouse model. What is encouraging is the clinical safety and efficacy of multispecific drugs targeting EGFR and HER3 in other malignancies such as head and neck cancer and colorectal cancer (clinical trials.gov identification numbers NCT01577173, NCT01652482).

In another study, dual blockade of EGFR and HER3 can increase the sensitivity of TNBC cells to phosphatidylinositol 3-kinase (PI3K) inhibitors, so it is necessary to conduct further research on the combination therapy of TNBC. Interestingly, a similar trend was observed with the EGFR×Notch bispecific antibody, which enhanced the therapeutic response of TNBC cells to PI3K inhibition, as evidenced by the significant reduction in the TNBC-CSC population [44]. Recently, a new strategy to provide antibodies to treat TNBC tumors has been developed, including the use of lipid-encapsulated calcium phosphate nanoparticles (LCP nanoparticles).

In this design, LCP NPs coated with polyethylene glycol (PEG) residues are able to bind to the anti-PEG Fab region of a bispecific antibody covalently linked to the anti-EGFR single-chain antibody domain.

LCP nanoparticles are not only functionalized with PEG×EGFR bispecific antibodies on the outer surface, but also loaded with cell death siRNA and a photothermal agent called indocyanine green inside the nanoparticles. Therefore, LCP NP functionalized with PEG×EGF bispecific antibody was effectively delivered to EGFR-expressing TNBC tumors, and after application of near-infrared radiation, TNBC cell apoptosis was induced in vitro and eliminated in in vivo mouse models TNBC tumor. This study provides a POC for bispecific antibodies to be used in the treatment of TNBC tumors on a nanoparticle platform based on gene therapy/photothermal therapy.

New TNBC target for bispecific antibodies

So far, many TNBC targets have been incorporated into the immune cell redirection bispecific antibody structure, including Trop2, CEACAM5, EphA10, P-cadherin, EpCAM, EGFR and mesothelin, and have been studied with immune checkpoint inhibitors Combination therapy.

In addition, bispecific antibodies that also target receptors on TNBC cells, including EGFR, HER3 and Notch, are also being evaluated recently.

In the field of cancer immunotherapy, the momentum of bispecific antibody therapy for TNBC treatment is continuing to increase. Some TNBC targets have become potential candidates for the development of bispecific antibodies in the future. In recent years, androgen receptor (AR) and cathepsin D have become targets of TNBC.

The expression of tumor-associated mucin 1 (MUC1) has recently been recognized as a biomarker of poor prognosis for TNBC. MUC1 has also been shown to contribute to the immune escape of TNBC.

A phase II clinical trial is underway to evaluate the efficacy of activated cytokine-induced killer cells (CIK) carrying CD3×MUC1 bispecific antibodies in the treatment of advanced breast cancer (ClinicalTrials.gov identification number NCT03524261). Quantitative plasma proteomic analysis showed that transforming growth factor β (TGFβ) signal is related to TNBC, which can predict tumor progression.

Several treatments for TGFβ in TNBC are being studied clinically, including anti-TGFβ/PD-L1 bifunctional fusion protein (ClinicalTrials.gov identification number NCT03579472). Heparin sulfate proteoglycan synthesizes decyl-1 (CD138) and γ-aminobutyric acid a (GABA) chloride channel subunits, called γ-aminobutyric acid receptor pi subunit (GABRP), which are two other proteins, Become a promising TNBC target.

Combining syndecan-1 or GABRP into the construction of bispecific antibodies for the treatment of TNBC is a feasible way to be explored.

Conclusion:

TNBC is a very aggressive subtype of breast cancer, and the medical needs for targeted therapy have not been met. Bispecific antibodies, especially CD3 T cell recipients, have become candidates for immunotherapy of TNBC.

Promising preclinical data on TNBC’s bispecific antibodies have been released, and several start-up pharmaceutical and biotech companies have recently received funding to continue this exciting research and bring these therapies to the clinic.

In fact, about two-thirds of Amgen’s pipeline molecules extended to the first stage are multispecific drugs, and BiTEs are the forefront of their innovation.

However, the transformation of TNBC bispecific antibodies from the test bed to the bedside is related to inherent challenges, and there are also some unresolved issues. For example, what preclinical model should be used to evaluate the safety and effectiveness of bispecific antibody therapy? Can these pre-clinical models effectively reproduce human physiology and realize the transition from the operating table to the bedside to the clinic?

Can biomarkers be used to predict the response of TNBC patients to slow-acting cancer immunotherapy? Can the next generation of TNBC targeting trispecific antibodies be engineered to reduce the cytokine storm associated with immune cell redirection, thereby broadening the therapeutic index? How to adjust immunosuppressive TME to improve the efficacy of TNBC targeting bispecific antibodies? …….

The bispecific CD3 T cell adaptor is designed to redirect the pan-T cell population to the tumor. Although the recruitment of CD8+ cytotoxic T lymphocytes (CTL) to tumors helps to initiate an effective anti-tumor immune response, re-aggregation of unrelated T cell subsets (such as depleted T cells) or immunosuppressive T cells should be avoided Recruitment of populations such as regulatory T cells (Treg).

In fact, it was recently discovered that the frequency of Treg cells in the peripheral blood of B-ALL patients who did not respond to blinatumomab treatment (CD3×cd19fda approved BiTE) was increased. Instead of recruiting and activating endogenous polyclonal T cell populations into tumor cells through CD3 complexes, it is better to consider recruiting T cells more selectively in the future bispecific antibody development of TNBC.

Another challenge associated with the redirection of bispecific T cells is cytokine release syndrome (CRS), a systemic inflammatory response caused by the rapid release of cytokines from immune cells. Although chimeric antigen receptor (CAR) T cell therapy is a more common problem, CRS has been prominently described in a phase I clinical trial involving superantigen antibodies and T cell stimulating factor TGN1412.

Efforts to control the cytokine storm through combination therapy or antibody engineering are underway, and TNBC targeting T cell binding bispecific molecules should be considered.

TME presents an additional barrier that may affect the effectiveness of bispecific antibiotics in TNBC. Tumor-associated macrophages, myeloid-derived suppressor cells, tumor-associated fibroblasts, Treg cells, and immune checkpoint molecules (such as PD-1) are the main contributors to the immunosuppressive properties of TME.

The application of immune checkpoint inhibitors in cancer treatment Cancer immunotherapy is very popular, with more than 750 clinical trials targeting the PD-1/PD-L1 axis. In fact, the anti-PD-1 monoclonal antibody pembrolizumab is currently undergoing clinical trials with the FDA-approved CD3×CD19 blinatumomab to determine whether the combination therapy can increase the total effective rate of B-ALL patients (ClinicalTrials.gov identification number NCT03160079).

In the preclinical model of TNBC, anti-PD-1 treatment can enhance the effects of CD3×Trop2 and CD3×CEACAM5 bispecific antibodies, thus indicating the potential benefit of modulating immunosuppressive TME in TNBC. In addition, the safety and tolerability of EGFR×TGFβ bispecific antibody combined with pembrolizumab in the treatment of EGFR-driven TNBCs are being evaluated clinically (ClinicalTrials.gov identification number NCT04429542).

However, due to the inherent heterogeneity of TNBC, the benefits of this combination therapy in TNBC are not fully understood, and further research is needed. Discovering and validating biomarkers that can predict which patients will respond to specific treatments is an important part of precision medicine, which helps to clarify the most appropriate combination therapy for TNBC patients.

The development of the next-generation trispecific T cell adaptor is proceeding smoothly, which includes a T cell binding part and two tumor antigen binding domains. The trispecific T cell adaptor designed with AND gate logic requires the participation of two TAAs simultaneously to promote T cell recruitment and subsequent tumor cell killing, while the trispecific T cell adaptor endowed with OR gate logic can interact with either TAA can recruit T cells after binding.

Recently, a trispecific antibody targeting CD38×CD3×CD28 was designed to target T cells to hematological tumors while providing effective T cell costimulation. This complex multispecific antibody structure can be designed into countless different forms to solve a given biological problem. Future research in the TNBC field may involve the development of next-generation trispecific antibodies to overcome immunosuppressive TME, solve cytokine storms, or recruit specific T cell populations for TNBC tumors.

(source:internet, reference only)

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