November 29, 2021

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Challenges and Development Strategies of Double Tumor Antibodies.

Challenges and Development Strategies of Double Tumor Antibodies.


Challenges and Development Strategies of Double Tumor Antibodies.


So far, compared with traditional anti-cancer treatment strategies, immunotherapy is considered to be the most promising systemic tumor treatment method, and it plays an indispensable role in improving the therapeutic effect, especially for the treatment of refractory cancers. .

Emerging cancer immunotherapies include cancer vaccines, chimeric antigen receptor T cell ( CAR-T ) therapy, cytokine therapy, immune checkpoint inhibitors, and tumor-targeting monoclonal antibodies ( mAbs ).

Among them, monoclonal antibodies have become a key and effective treatment method in cancer treatment due to their ability to specifically target molecules.

However, due to the complex pathogenesis of tumors, monoclonal antibodies directed against a single target are often insufficient to show sufficient therapeutic effects.

Therefore, bispecific antibodies ( bsAbs ) against multiple targets emerged, and its development has changed the field of tumor immunotherapy.


Because it can target two epitopes in tumor cells or tumor microenvironment ( TME ) at the same time , bsAbs has gradually become an important and promising component of the next generation of therapeutic antibodies.

Most bsAbs currently under development are designed to form an artificial immune contact by closely linking immune cells, especially cytotoxic T cells, with tumor cells, ultimately leading to selective attack and lysis of targeted tumor cells.

Although there are many forms of bsAb, according to the presence or absence of FC fragments, it can be roughly divided into two categories: IgG-like and non-IgG-like. The presence of the Fc fragment significantly exerts an additional effector function.

Currently, many preclinical and clinical trials are underway. However, the application of bsAb drugs in tumor therapy still faces huge challenges, including tumor heterogeneity and mutational burden, uncontrollable tumor microenvironment, insufficient costimulatory signals to activate T cells, the necessity of continuous injection, and fatal Systemic side effects and off-target toxicity.

In response to these problems, this article proposes a variety of strategies to solve these problems, including preparing multispecific bsAbs, discovering new antigens, combining bsAbs with other anticancer drugs, developing bsAbs based on NK cells, and preparing bsAbs in situ.


bsAb’s design strategy

bsAbs exhibit dual specificity by binding to different antigens or epitopes at the same time. They have received extensive attention in the field of tumor therapy. There are four main functions:

(a) Redirect specific immune effector cells and selectively destroy cancer Cells;

(b) Targeting a variety of cell surface antigens to improve targeting specificity;

(c) Delivery of drugs into the tumor; and (d) Blocking two biological pathways to improve the efficacy and durability of treatment.


Among these functions, one of the most commonly used functions is to bring immune effector cells close to cancer cells, thereby reducing systemic toxicity and avoiding drug resistance. bsAbs are roughly divided into two groups: (a) IgG-like ( with Fc region ) and (b) ) Non-IgG-like ( no Fc region ).


Challenges and Development Strategies of Double Tumor Antibodies.


Due to its larger size and FcRn-mediated recycling process, the IgG-like bsAb with the Fc region has a longer circulating half-life than the bsAb without the Fc region. The purification is more convenient, and the solubility and stability are improved.

More importantly, it may have greater clinical therapeutic potential to retain a variety of Fc-mediated effector functions, including antibody-dependent cytotoxicity ( ADCC ), complement-dependent cytotoxicity ( CDC ) and Ab-dependent cell Phagocytosis.


In contrast, non-IgG-like bsAb fragments show relatively low circulation dynamics, but have better tissue penetration, lower immunogenicity, and lower non-specific activation of the innate immune system. This form of bsAb mainly depends on its antigen binding ability to perform multiple functions.


In order to extend the half-life of non-IgG-like structures while maintaining its original biological activity, safety and low immunogenicity, various strategies can be used to increase its molecular weight and extend its half-life in serum:

(a) Use peptide linkers Make Ab fragments form multimers;

(b) With other molecules such as human serum albumin, polyethylene glycol ( PEG ), carbohydrates, N-(2-hydroxypropyl) methacrylamide ( HPMA ) and dextran Sugar coupling.


The multimerization of Ab fragments, taking multimeric single-chain antibodies as an example, is a core strategy for non-IgG-like forms.


The development history of bsAb technology platform

The history of bsAb can be traced back to 1961, when Nisonoff and Rivers first proposed the concept of producing multispecific Abs by mixing different Ab fragments. In the early stages when hybridoma technology and chemical recombination methods were established in 1975 and 1985, respectively, the production of BSAb relied on the fusion of hybridoma somatic cells to produce four tumors or two different monoclonal antibodies F(ab′) 2 molecules. Heteroconjugate. However, since the chemical coupling may bsAb inactivation, expand or aggregation, further development is hindered bsAb .

At the same time, the quaternary technology often mistakenly assembles the heavy and light chains, resulting in a decrease in the yield of the required pairing and an unexpected immune response. Essentially, the correct heavy chain-light chain pairing usually includes two aspects: (a) the correct pairing of two different heavy chains; (b) the light chain is combined with the corresponding correct heavy chain.

These problems urgently require improved methods to generate correctly paired bsAbs. Initially, the problem of heavy chain/light chain mismatch was solved in 1995 by creating a chimeric mouse/mouse quadruple, which ensures the correct pairing of homologous heavy chains.

For example, the clinically approved catumaxomab is composed of mouse IgG2a anti-CD3 hapten and rat IgG2b anti-epithelial cell adhesion molecule ( EpCAM ) hapten. In 1998, the application of the same light chain to two heavy chains also showed an effect. However, heavy chain mismatch and immunogenicity still exist, the scope of clinical application is limited, and better solutions are needed.


The rapid development of genetic engineering technology provides an opportunity to overcome the above shortcomings, which promotes the development of the second wave of bsAb. The production of these newly developed bsAbs mainly relies on recombinant DNA technology, which can produce chimeric or humanized antibodies while controlling the size, affinity, bispecificity, half-life, stability, solubility, and biodistribution of the bsAb .

To meet the different needs of the required target products. In 1996, Ridgway et al. described the “knobs into holes” ( KiH ) method of using CH3 domain mutations to generate humanized anti-CD3×CD4 hybrids . This method is to replace a large amino acid ( hole ) with a small amino acid in one heavy chain of bsAb, and vice versa in the other heavy chain ( knob ) of bsAb , and finally guide the formation of a heterodimer according to the electrostatic orientation theory. Rather than homodimers.

Phage display technology was used to screen CH3 mutants that formed stable heterodimers. In addition, in order to reduce monomer or homodimer impurities, some researchers have further designed the CH3 domain of the heavy chain to add interchain disulfide bonds to further improve the heterodimer and provide the possibility of purification.

This technology can also produce various types of heterodimeric proteins by fusing peptides, protein ligands or Ab fragments to both ends of the Fc chain. Later, the emergence of a new technology called CrossMab further reduced the by-products of heavy chain/light chain pairing.

This is achieved by exchanging the heavy chain and light chain domains in the Fab region of an Abs, which results in a change in the molecular structure of the interface between VH-VL and CH1-CL, and a complete Fc and antigen binding domain is constructed. The stability and affinity of CrossMab-bsAb against Angiopoietin-2 and Vascular Endothelial Growth Factor a are higher than its parent antibody.

CrossMab form can be further divided into three subtypes, CH1 with CL ( CrossMabCH1-CL ) , VH with VL ( CrossMabVH-VL)), or VL-CL with VH-CH1 ( CrossMabFab ). CrossMab does not require sequence optimization or additional linkers, making it an attractive method for designing new bsAbs. In addition, it can be combined with KiH to ensure the correct pairing of the heavy chain.


With the advancement of genetic engineering technology and the emergence of multiple methods such as phage display, protein engineering, and transgenic mice, more than 100 forms of bsAb have been developed. These newly developed forms circumvent previous manufacturing problems such as instability, low yield, and immunogenicity, thereby accelerating the speed from the test bench to the clinic.

As mentioned above, the general bsAb format can be simply divided into two categories. On the one hand, in addition to quadromas, KiHs and CrossMab, IgG-like forms also include dual variable domain Ig ( DVD-Ig ), IgG single-chain Fv ( IgG-scFv ), two-in-one or dual-acting Fab antibody ( DAF ) and κλ-body and other forms.


On the other hand, non-IgG-like forms include single-chain antibodies, nanobodies, dock and lock (DNL) methods, and other multivalent molecules. Single-chain antibody refers to the combination of two variable region domains from the parent Ab light chain and heavy chain, namely VH and VL. They are connected by a peptide linker, usually a (G4S) 3 sequence. Single-chain antibody-based formats include scFvs, sdAb, ta-scFv, diabodies, TandAbs, and DART.

In this format, BiTE is the most typical and widely used one. The United States Food and Drug Administration ( FDA ) and the European Medicines Agency ( EMA ) approved anti-CD19×CD3 blinatumomab ( Blincyto; Amgen ) in December 2014 and December 2015, respectively , for the treatment of chromosome-negative Philadelphia relapses Or refractory precursor B-cell acute lymphoblastic leukemia ( B-ALLs ). So far, various bsAb forms have been summarized in many other reviews.


Clinical application of bsAb


The first clinical trial of bsAb was conducted in 1990. Use chemically coupled anti-CD3 mAb OKT3 and anti-glioma mAb NE150 to treat malignant glioma.

In 1995, bsAb was the first to treat chemotherapy-resistant non-Hodgkin’s lymphoma ( NHL ) by intravenous injection of anti-CD3×CD19 bsAb . Disappointingly, although limited systemic toxicity was observed, there was no clinical response in this test, only an increase in serum tumor necrosis factor ( TNF-α ) and CD8+ T cells was observed .

In the same year, a BiTE first, two- th targeting CD3 and 17-1A single-chain antibody even connected together, i.e. anti blinatumab precursor of the CD19 × CD3.

In 1997, bsAbs targeting FcγRIII (CD16) and Hodgkin-related antigen CD30 to activate NK cells showed encouraging anti-tumor activity in clinical phase I/II studies.

Using recombinant DNA technology, blinatumomab avoids the previous problems of low yield, unclear by-products and complicated purification procedures.

One year later, Blinatumomab entered the first phase of human research in Germany and Sweden, in which 21 patients with relapsed or refractory NHL received short-term intravenous injections.

Until 2004, NHL patients was first observed b Lina t UMO m ab clinical response meaningful daily dose of 15μg / m2. A few years later, blinatumomab was used in extensive clinical trials until it was finally approved by the FDA and EMA. In the following years, the treatment range of blinatumomab was further broadened.

In 2018, with FDA approval for the treatment of pre-B ALL patients with MRD ≥0.1% after the first or second complete remission, The bsAb field has experienced explosive growth, and the future clinical development prospects are broad.


Challenges and Development Strategies of Double Tumor Antibodies.


Solution for antigenic mutation escape

In order to process two targets simultaneously while maintaining their potency, the first step in generating bsAbs is to determine the appropriate target antigen. Similar to traditional Abs, the targets of bsab should meet the following criteria:

(a) They are clearly expressed in target cells rather than adjacent normal cells to avoid non-specific toxicity ( also known as “targeted non-tumor” toxicity) );

(b) They are closely related to the phenotype of malignant tumors or the signal pathways that prevent immune tolerance caused by antigen mutations.


Currently, only a small portion of tumor-associated antigens ( TAA ) are identified and strictly meet the above criteria, which is frustrating.

Among them, CD19 is the most representative. CD19 is expressed on most B-ALLs and is essential for the development and function of B cells. Therefore, it is an important target for CAR-T cells or bsAb-based immunotherapy.

However, it has been reported that the failure of CD19-specific CAR-T cells or anti-CD19×CD3 BiTE in B-ALL treatment is attributed to the gene mutation of CD19, which leads to the loss of CD19 extracellular domain, conformational changes, and cellular changes.

Impaired surface transport, or phenotypic transition from ALL to acute myeloid leukemia ( AML ). Therefore, if only one TAA targets cancer cells, the genetic modification of the selected TAA may pose a threat to the effect of immunotherapy, which brings us a huge challenge.


Strategy 1:

Develop multi-specific antibodies ; to avoid immune invalidity related to a single target, some researchers have proposed the idea of ​​multi-target antibodies to simultaneously recognize multiple antigens on the surface of target cancer cells to improve affinity.

For example, the three members of the erythropoietin-producing hepatocyte ( Eph ) receptor family, EphA2, EphA4, and EphB4, are involved in the progression and metastasis of many malignant tumors, and they are all attractive targets for anti-tumor therapy.

On this basis, a trispecific antibody was designed by connecting the anti-EphB4/EphA4 diabody to the C-terminus of the intact anti-EphA2 antibody. Another example of this strategy is a dual-targeted single-chain antibody trispecific antibody that simultaneously recognizes CD123 and CD33 on AML cancer cells. Compared with single-targeted drugs, it shows significantly stronger anti-tumor activity.

In addition, the four-specific Abs targeting endothelial growth factor receptor ( EGFR ), HER2, HER3 and VEGF also show more effective anti-tumor effects than single-targeted drugs in vitro and in vivo, and can destroy the parental bsAb induced Resistance.


Strategy 2:

Discovery of neoantigens; due to its high mutation load and obvious resistance to current tumor immunotherapy, it is a challenge to identify neoantigens and determine the relevant internal mechanisms to pave the way to improve anti-tumor strategies, but it is also Urgent task.

In short, neoantigens, also known as tumor-specific antigens (TSA), are derived from non-synchronous somatic mutations and are specifically expressed in tumor cells, but are completely absent in normal cells. Previously, most immunotherapies targeted TAAs because they were usually overexpressed in a group of tumor types and therefore covered a wider patient population, such as CD19.

However, cumulative studies based on TAA treatment have reported potential damage to normal tissues and the clinical efficacy is not ideal.

On the contrary, TSAs are selectively expressed in tumor cells, and there are differences between different individuals, thereby providing personalized opportunities for TSA-dependent immunotherapy.


Generally speaking, the selection of Ab targets can be roughly divided into three generations:

(a) The first generation consists of “validated antigens”, which have been confirmed by extensive experiments and clinical trials;

(b) The second generation includes modified peptides , Which means that it is either a different epitope from the “verification antigen” or the same epitope with improved properties;

(c) The third generation includes those new ones selected based on genomics, proteomics, or cell-based functional strategies.


Antigen found. Obviously, the recognition of TSAs is closely related to the third-generation Ab targets. Technically speaking, the combination of whole-exome sequencing and fast-developing software algorithms is currently the most interesting and promising method for identifying neoantigens.

Whole-exome sequencing is the most important kind of genome-related technology, including obtaining DNA samples, breaking them into fragments, screening and extracting coding fragments, amplifying fragments, sequencing fragments, and finally analyzing the results with reference genomes.

According to the principle of DNA codon pairing, the mutated DNA sequence is converted into the amino acid alternation in the antigen. Subsequently, various algorithms can be used to predict neoantigens with high binding affinity to MHC molecules.

The cell-based method uses whole cancer cells as a platform to selectively generate corresponding monoclonal antibodies. Antigens with high binding activity to the surface antigens of cancer cells are screened out.


Combination strategies to avoid T cell anergy immunotherapy


In the process of host immune surveillance, T cells are important sentinel cells that antigen-presenting cells present MHC peptide complexes to T cell receptors ( TCR ), provide secondary costimulatory signals to activate primitive T cells, and promote effector T cell proliferation.

On the contrary, if costimulatory molecules are missing or replaced by co-inhibitory molecules, that is, immune checkpoints, T cell function is inactivated or incompetent, so that tumor cells cannot be specifically eliminated.

Among the many immune checkpoints, programmed death receptor 1 ( PD-1, also known as CD279 ) is the most important and widely studied molecule.

It is expressed on T cells of TME and interacts with tumor cells or tumor infiltrating lymphocytes with its ligand PD-L1 to mediate tumor immunosuppression.

At the same time, it has been observed that the lack of clinical efficacy of bsAb-dependent treatment may be partly due to immunosuppression in TME, especially through the PD-1/PD-L1 pathway.

For example, a study of anti-AC133×mCD3 bsAbs in diabody format reported that radiotherapy induced the apoptosis of tumor infiltrating lymphocytes ( TIL ) and subsequent tumor growth, which was mediated by the PD-1 pathway.

Therefore, additional PD-1 blockers are very beneficial to increase the number of TIL, restore anti-tumor immunity and improve survival.

In general, the combination strategy mainly includes immune checkpoint blocking, CAR-T cells and other strategies so far.


Strategy 1:

Simultaneous application of BSAB and immune checkpoint inhibitors to suppress monoclonal antibodies; the application of immune checkpoint inhibitors has promoted the development of T cell synergistic anti-tumor immunotherapy, especially the combined application to recruit and mobilize T cells to fight cancer.

In one study, the application of anti-CD33×CD3 BiTE ( AMG330 ) in a mouse model resulted in the up-regulation of tumor cells PD-1 and showed significantly impaired T cell-mediated tumor cell lysis. In contrast, blocking the PD1/PD-L1 interaction enhanced AMG330-mediated cell lysis.

In another study, dual administration of anti-PD-1 and anti-PD-L1 monoclonal antibodies before anti-CEA×CD3 BiTE can effectively block the immunosuppression in TME and maximize the cytotoxicity of effector T cells change.

In clinical trials, the combined application of bsAb and immune checkpoint inhibitors can also work synergistically to improve the anti-tumor effect and patient survival. In addition, in previous drug-resistant HER2+ breast cancer treatment studies, anti-tumor effects were observed when HER2-TDB ( a trastuzumab-based T cell recruitment bsAb ) and PD-L1 inhibitors were used.

These benefits include enhanced tumor growth inhibition and enhanced response durability.

Recently, a new type of trivalent T cell redirection bsAb was constructed by the DNL method, namely, (E1)-3s and (14)-3s, targeting Trop-2 and CEACAM5, respectively, and combined with PD-L1 blockers, in vitro and Both showed enhanced anti-tumor activity in vivo.


Strategy 2:

bsAb that targets immune checkpoints; this is another attractive option that uses both immune checkpoint blockade and bsAb technology. The result is to design bsAbs that target both immune checkpoints and tumor antigens at the same time. Strategically superior to the combination of immune checkpoint inhibitors and bsAb.

In terms of mechanism, this is achieved by binding to PD-1/PD-L1 blockers in TAA-positive cancer cells. For example, an anti-PD-L1×EGFR bsAb to activate PD-L1 blockers in EGFR overexpressing tumor cells.

It has a symmetrical tetravalent taFv-Fc structure with an Fc domain to mediate related functions. It has been confirmed that anti-PDL1×EGFR-bsAb can enhance the accumulation of Ab at tumor sites and prevent serious systemic autoimmune-related adverse events in a variety of epithelial malignancies (such as colorectal cancer and non-small cell lung cancer).

Similarly, some researchers have designed two new bsAbs: anti-PD-1×c-Met DVD-Ig and IgG single-chain antibody, both of which exhibit effective anti-tumor immune activity in vitro and in vivo. Similarly, when another bsAb targeting PD-L1 and chondroitin sulfate proteoglycan 4 (CSPG4) is used to treat melanoma and other CSPG4+ malignancies , while maintaining the safety of PD-1/PD-L1, it also has Help improve the efficacy and selectivity of anti-tumor.

In addition, using anti-CD33×CD3 BiTE as a scaffold, the extracellular region of PD-1 is fused to the scaffold to generate a trispecific antibody, recruit T cells to CD33+AML cells, and block PD-1 Immune checkpoint pathway.


Although the current joint research on bsAb and immune checkpoint blockade mostly focuses on the PD-1/PD-L1 pathway, other immune checkpoints, such as cytotoxic T lymphocyte-associated protein 4 ( CTLA-4, CD152 ) and T Cellular Ig Mucin 3 (T IM-3 ) also plays an indispensable role in immunosuppressive TME.

In order to further reduce the peripheral toxicity and only block the immune checkpoint in the tumor area, a new set of bsAbs are being creatively designed to target two different immune checkpoints at the same time, such as the anti-PD-1/TIM-3 in CrossMab format. bsAb, or targeted checkpoint inhibitor and T cell co-stimulatory receptor, in the form of IgG1 anti-CTLA-4×OX40bsAb ( named ATOR 1015 ).


Strategy 3:

Combine CAR-T cell therapy to provide costimulatory signals ; in addition to the above T cell immunosuppressive signals, in the absence of costimulatory signals, continuous stimulation of the TCR-CD3 signal pathway through CD28 or 4-1BB (CD137) molecules is Another important explanation for effector T cell incompetence or apoptosis.

Initially, this problem was solved by using anti-4-1BB monoclonal antibodies or the extracellular domain of 4-1BBL or anti-CD28 monoclonal antibodies as adjuvants to assist bsAb therapy, showing a prolonged activation time of effector T cells.

In addition, by fusing two members of the tumor necrosis factor superfamily (including CD40L, CD27L, OX40L and 4-1BBL) into a T cell relocation diabody, T cell stimulation and anti-tumor activity are finally enhanced. However, these solutions show that the duration of effector T cells is relatively short.

To solve this problem, a group of researchers proposed a new type of T cell targeting complex to eradicate AML cells. The complex includes an anti-CD3×peptide epitope (E5B9) bsAb, which is a universal effector module and A target module consisting of an anti-CD33 single-chain antibody fused to a peptide epitope (E5B9) and an extracellular domain of 4-1BBL.

Compared with the traditional anti-CD33×CD3 bsAb, the complex not only has higher efficacy in activating T cells and inducing CD33+ tumor cell lysis, but also enhances the killing effect on CD33 low-expressing cells through additional costimulatory signals. More importantly, this novel and flexible modular system can enhance the anti-tumor function for a long time and has a wide range of application potential in tumors.

In addition, in order to provide long-acting effector T cell functions, other researchers have developed an optimization strategy that combines the bsAb strategy with the CAR-T cell strategy to produce a new set of bsAbs . This new bsAb, named frBsAb, is a chemical heteroconjugate of two monoclonal antibodies, fused with folate receptor (FR) and the selected TAA.

Correspondingly, the transformed T cell is called BsAb-IR28z, and its extracellular FR domain is connected to the CD8α-hinge and transmembrane region; the intracellular CD3z part is fused with CD28.

Therefore, when frBsAbs treat tumor cells ( through the recognition of TAA)) When cross-linked to mediated T cells, they not only induce transient T cell activation, but also prevent incompetent or antigen-induced cell death by simultaneously stimulating the costimulatory molecule CD28, and ultimately exert enhanced anti-tumor ability.

Among costimulatory molecules, the combination of CAR-T cells and bsAbs also provides many advantages, and is being developed as a promising method for the treatment of malignant tumors.

Recently, people have designed a single gene modified T cell product CAR-T-EGFRvIII for the treatment of glioblastoma multiforme (GBM).

BiTE-EGFR cells secrete additional anti-EGFR×CD3, while maintaining the traditional CAR-T cytoskeleton, and express EGFRvIII on the surface of T cells.

Since EGFR and EGFRvIII are both key targets of GBM tumor cells, this innovative invention has advantages over CAR-T cells and bsAb as a monotherapy because it not only confirms the effective anti-tumor response of antigen-positive tumor subtypes, but also Moreover, it is relatively safe for healthy tissues and avoids the disadvantages of its parental technology, such as tumor antigen heterogeneity, non-specific toxicity related to systemic application, the necessity of continuous injection, immunosuppression, T cell failure, etc.


Essentially, so far, a group of innovative universal or modular CAR- ( modCAR- ) T cells and their respective adaptor molecules ( CAR adaptors ) have been used to circumvent some fatal side effects of traditional CAR-T cells.

This strategy divides TAA recognition and T cell activation into two parts, and relies on the CAR adapter as a bridge to relocate modCAR-T cells to multiple TAAs, and use the same modCAR-T cells to target multiple tumor types.

In this way, the function of effector T cells can be fine-tuned without eliminating them. Currently, CAR adapters can be divided into several subtypes based on their structure, including small molecules (such as folic acid and fluorescein isothiocyanate), monovalent and bivalent nanobodies, single-chain antibodies, Fabs, and Abs.


Bispecific strategy and NK cells

So far, most attempts to enhance anti-tumor immunity have focused on enhancing T cell responses through CAR-T cells or bsAbs, especially after the success of blinatumomab. However, it is worth noting that this kind of T cell targeted therapy is limited by some side effects, the most challenging of which is the fatal cytokine release syndrome ( CRS ).

Although great efforts have been made to reverse CRS by blocking IL-6 or inhibiting monocyte/macrophage activation, achieving optimal toxicity management while maintaining full therapeutic potential remains a challenge. In addition, since most of the T cell relocation targeting bsAb is aimed at combating hematological malignancies, the treatment of solid tumors mediated by bsAb needs further research.

NK cells, as the front-line fighters of natural immunity and anti-tumor, have also attracted widespread attention in the past few decades.

In humans, various studies have shown that the damage or loss of NK cell function is closely related to the high risk of tumor occurrence, development and metastasis.

It is worth noting that a high proportion of NK cells infiltrating solid tumors is associated with better clinical efficacy, which has been confirmed in colorectal cancer, breast cancer, clear cell renal cancer, head and neck cancer, and pharyngeal cancer. Therefore, targeting NK cells through immunotherapy is an attractive anti-tumor strategy.


In terms of mechanism, activated NK cells can eliminate tumor cells through three direct or indirect strategies:

(a) release particles, such as secreted lysosomes, which contain perforin and granzyme, which induce cell membrane lysis or apoptosis;

(b) Through the interaction of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand on tumor cells to activate the target cell caspase;

(c) secrete a variety of factors to regulate the functions of other immune cells, and indirectly kill tumor cells. Based on these theories, NK cell adapters (NKCEs) and CAR-NK cells are designed to use NK cell-mediated cytotoxicity to fight tumors.


NKCE is constructed by connecting anti-NK cell receptors ( mainly CD16 ) and one TAA ( bispecific killer engagers, BiKEs ) or two TAA ( trispecific killer engagers, TriKEs ) single-chain antibodies. So far, anti-CD16 BiTEs have been effectively used to target a wide range of TAAs, including CD19, CD20, CD30, CD33, CD133 and EPCAM.


The anti-CD30 × CD16A BIKE ( AFM13 ) the treatment of relapsed or refractory CD30 + Hodgkin tumor or NHL, I / Phase II clinical trials have been published. Interestingly, in a study of myelodysplastic syndrome ( MDS ), BiKE against CD16×CD33 can not only eradicate CD33+MDS cells, but also target CD33+ myeloid-derived suppressor cells to reverse the TME Immunosuppression, improve the efficacy of anti-tumor.

In contrast, with additional single-chain antibodies, TriKEs gain more benefits by targeting more taa or acting on more NK cell activation receptor targets.

Recently, a remarkable trifunctional NKCE was reported, which interacts with CD16 and NKp46 ( a natural cytotoxic receptor (NCR) ) on NK cells and TAA on tumor cells.

In mouse models of aggressive and solid B-cell lymphoma, this cleverly designed TriKE leads to the full activation and enhancement of NK cells’ cytotoxicity to tumor cells , and has been shown to be more effective than BiKE, which activates CD16 and NKp46, respectively. Anti-tumor function.

In addition, another design adds IL-15 cross-linking agent, upgrades the anti-CD16×CD33 BiKE to TriKE, because NK cells self-sustained proliferation, thereby improving tumor cell killing efficiency.

It is worth noting that TriKE ( GTB-3550) against CD16×IL-15×CD33) Single-center phase I/II clinical trials are already in progress for the treatment of CD33+ high-risk MDS, refractory/relapsed AML, or advanced systemic mastocytosis.

In addition to superior anti-tumor effects, BiKE and TriKE also have lower toxicity and higher safety than T cell adaptors, which means that the risk of CRS or non-targeted cytotoxicity is lower, and its application in solid tumors The prospects are broader. Similar to NKCE, mature NK cells can also be used as an interesting candidate cell to express CARs against abundant TAAs such as CD19, CD20, CD244 and HER2.


In situ bsAb


Despite the great success of bsAb, the therapeutic potential of exogenous administration is hindered by short cycle kinetics and targeted non-tumor toxicity.

Therefore, the researchers proposed the concept of generating bsAb in situ to overcome immunosuppressive TME and avoid continuous drug infusion.

At present, the methods for preparing bsAb in tumor tissues mainly include engineered oncolytic viruses ( OVs ), transfection of T cells, and transfection of mesenchymal stem cells (MSCs) .


In 2015, the FDA approved Imlygic, the transgenic oncolytic HSV therapy talimogene laherparepvec ( T-VEC ), and expresses GM-CSF for the treatment of advanced melanoma. This represents a milestone for OVs and has facilitated a large number of preclinical studies and clinical trials.

Due to the advantages of tumor-specific expression and virus-mediated T-cell recruitment, OVs are an attractive bsAb delivery platform. Currently, these OVs carrying BiTEs are undergoing pre-clinical evaluation: EphA2-T cell vaccine virus producing anti-CD3×EphA2 BiTE; adenovirus anti-EGFR×CD3 BiTE ( ICOVIR-15K ); anti-CD3×CEA/CD20 BiTE measles Virus ( MV-BiTE ); Anti-EpCAM×CD3 BiTE engineered oncolytic group B adenovirus Enadenoutucirev ( EnAd ).

The first OV to carry BiTE was a double-cleared vaccinia virus ( western reverse Straine), which is designed to encode EphA2 targeting BiTE. In the A549 lung cancer xenograft model, the addition of exogenous IL2 can increase the activation and proliferation of T cells in vitro, and increase the survival rate.

Later, the researchers modified the adenovirus ICOVIR-15K to produce anti-EGFR×CD3 BiTE, which successfully promoted T cell proliferation without increasing IL-2. Another similar structure, MV-BiTE, provides continuous immune protection for mice with normal immune function and solid tumor xenograft models, and promotes long-term tumor regression without recurrence.

EnAd carrying anti-EpCAM×CD3 BiTE further showed the ability to kill target cells, while overcoming the immunosuppressive TME in human primary malignant ascites samples.

In general, OVs engineered to produce BiTE can attack and kill cancer cells by non-specific direct oncolysis or infection of antigen-positive cancer cells, and then the replication-dependent expression of BiTE can activate endogenous CD4+ and CD8 + T cells, thereby causing immune-mediated cytotoxicity to these cancer cells. This improved oncolytic virus therapy has bright prospects because it is superior to traditional OVs or bsAbs:

(A) biTEs derived from modified OVs selectively target antigen-positive cancer cells without relying on MHC/TCR molecules Presents or other costimulatory signals, so even when cancer cells lose MHC expression, it can also increase the effectiveness of anti-tumor T cell responses and stimulate immunity.

(B) Under the control of the late virus promoter, by limiting the continuous BiTE production of virus-infected cancer cells, not only the system toxicity is minimized, but the concentration of BiTE in TME is greatly increased, and the short half-life of BiTE is also solved . problem.

(C) OVs mainly infect and replicate in cancer cells, and spread between cells, reducing the damage of adjacent healthy tissues.

(D) BiTEIt can target virus-infected cancer cells or antigen-positive cancer cells that are not infected.

(E) Modified OVs can stimulate CD4+ and CD8+ T cell-mediated immunity while avoiding immunosuppression.

(F) The method has high safety, small side effects and convenient application.


In addition, similar to the modified OVs targeting tumor antigens, OVs carrying BiTEs targeting mesenchymal cell antigens have a better curative effect on refractory and stromal-rich tumors.

Encoding genetically engineered enzymes targeting fibroblast activation protein ( FAP ) can not only kill infected and replicated tumor cells, but also use the subsequently produced anti-FAP BiTE to activate T cells and selectively deplete FAP+ Cancer-associated fibroblasts ( CAFs ).

They eventually reverse the CAF-mediated immune suppression, break the interstitial barrier, allow OVs to penetrate into the tumor site, and cooperate to restore TME to an immune response state.


Transfection of autologous tumor-specific T cells ; At present, transfected autologous tumor-specific T cells have attracted attention because of the theoretical combination of CAR-T cells and bsAb and overcoming the limitations of the two.

Nearly ten years ago, when human peripheral blood lymphocytes, especially CD3+ T cells, were transfected with an engineered HIV-1 based lentiviral vector to produce cells against CEA×CD3 diabody in the body, the lymphocytes were first It is believed to be the carrier of bsAb.

Recently, this has been demonstrated by CD123-ENG T cells, which are transduced by a retroviral vector encoding a bispecific adaptor molecule that targets CD123+ tumor cells of AML and CD3+ T cells.

In terms of mechanism, ENG T cells not only recognize and kill CD123+ tumor cells, but also redirect and activate unmodified bystander T cells to the tumor site through antigen-dependent BiTE secretion, which synergizes to produce a powerful anti-tumor immune response .

In addition, in order to avoid accidental and excessive killing of normal hematopoietic stem and progenitor cells, CD20.CD123-ENG T cells are designed to contain the suicide gene CD20, which allows selective depletion in the presence of rituximab and complement.

Other similar ENG T cells include EphA2-ENG T cells and CD19-ENG T cells. In addition, some researchers have constructed CD19-BiTE-transferred T cells through mRNA electroporation technology and rapid T cell expansion protocol.

In vitro and in the invasive Nalm6 leukemia mouse model, compared with CAR RNA-transfected T cells, Its anti-tumor immunity has also been enhanced. Transgenic T cells show obvious advantages, because once activated, they will produce enhanced BiTE and redirect the resident T cells to the tumor site.

In order to further improve the anti-tumor efficiency, the costimulatory molecules CD28 and/or 4-1BBL are introduced into the surface of ENG T cells, namely CD19-ENG.4-1BBL/CD80, which significantly increases the antigen-dependent cytokines ( IL-2 and IFNγ ). Produce and promote the proliferation of T cells, and finally show the best response in vivo and in vitro.


Transfection between mesenchymal stem cells ( mesenchymal cells STEM, of MSCs ) in addition to tumor cells and T cells, the MSC may be used as a cell carrier, stable expression of the bsAb situ. Bone marrow mesenchymal stem cells have the unique advantages of low immunogenicity, tendency to migrate to tumor sites, ability to track microscopic metastasis, and easy transduction through viral vectors.

In a study, an immortalized human MSC line ( SCP-1 ) was genetically modified through a lentiviral vector to produce a fully humanized anti-CD33×CD3 bsAb to increase the expression of the costimulatory molecule 4-1BBL. The enhanced anti-antigen-specific T cell response makes MSCs an effective transmitter of bsAb targeting tumors, thereby prolonging tumor regression time.

Another study used transfected human umbilical cord-derived MSCs to secrete anti-CD19×CD3 TandAbs, and used the indoleamine 2,3-dioxygenase ( IDO ) pathway inhibitor, D-1MT, to prove that this combination is one A promising treatment strategy.

In addition, E1A-modified MSCs can also be designed as viral transporters and amplifiers to release engineered adenovirus and re-infect tumor cells after reaching the tumor site or metastasis.

They secrete bsAb and activate T cell responses to tumors. This is a dual virus-loaded MSC ( named MSC.CD3-HAC.E1A ) carrying the bifunctional fusion protein CD3-HAC, composed of anti-CD3 single-chain antibody and high affinity consensus ( HAC ) PD-1, which ultimately promotes tumor elimination And reverse the immune tolerance of TME.


Minicircle ( MC ); MC is another promising alternative for the production of bsAb in the body. It is a group of non-viral DNA carriers that can continuously express the transgene product at a high level after delivering the transgene product to the mouse liver through a hydrodynamic process.

Some researchers used MC to design MC-bsAb to generate anti-CD20×CD3-bsAb, which mediates T cells to kill human B-cell lymphoma cells in vitro and in xenograft mouse models.

This method is very attractive because it is relatively stable, inexpensive, and can maintain the therapeutic concentration of bsAb in the blood circulation for several weeks or even longer.




In the past 30 years, people have witnessed a huge transformation, from just the development and modification of basic Abs to more complex Ab derivatives, with various shapes and sizes, especially bsAb.

The bsAb technology has extraordinary prospects in clinical applications, has attracted the attention of researchers and has developed extremely rich forms, laying a solid foundation for BSAB-based cancer immunotherapy. As of September 20, 2019, a total of 183 clinical trials of bsAb ( mostly in the field of cancer ) have been published on the NCBI’s official website .

However, despite the various strategies mentioned above, the process of commercial production of bsAb drugs is still hindered by various obstacles.

More specifically, the manufacture of bsAb is time-consuming and expensive. It requires suitable, safe and economical cell line production, procedures, analysis and purification methods to obtain the desired product.

In addition, a series of problems after Ab production, including but not limited to the degradation, aggregation, denaturation, fragmentation and oxidation of Abs, must be resolved before being given to patients.

At the same time, more clinical trials are needed to explore the best route of administration and the best dose to increase the concentration of target tissues, reduce systemic side effects, and even control-release preparations.


Overall, it is estimated that most bsAbs ( 67% ) in current clinical trials are aimed at combating hematological malignancies. In contrast, bsAb targeting solid tumors is worthy of further study, because it has unavoidable adverse effects on normal tissues or other complex factors ( including immune tolerance cancer stroma, neovascular disorders, and insufficient penetration of bsAb drugs ) .

Therefore, people are enthusiastic about ongoing research on bsAbs in solid tumors. These studies are expected to produce promising results in the near future, although the conversion of bsAbs into clinically applicable drugs may be time-consuming and require a lot of effort.

In short,The results of the bsAb study proved the promise of these molecules in new drug design and subsequent clinical applications in cancer treatment.




Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020 May;17(5):451-461.

Challenges and Development Strategies of Double Tumor Antibodies.

(source:internet, reference only)

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