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Over the past 30 years, traditional monoclonal antibodies have occupied an important position in the development of therapeutic biomolecule drugs. In 1993, Belgian scientists published a paper in Nature, discovering for the first time in the alpaca body a naturally occurring variable region of heavy-chain antibody (HcAb) lacking light chain with a molecular weight of only 15 kDa, which is 1/10 of the molecular weight of traditional antibodies.  It is also the smallest known natural antibody, and it is named a single domain antibody (sdAb).

Due to its unique advantages of a small structural domain, good stability, flexible route of administration, high solubility, and microbial expression, sdAb has gradually become an emerging force in the new generation of therapeutic biopharmaceuticals and clinical diagnostic reagents. In addition, sdAb is more likely to recognize antigens that cannot be captured by traditional antibodies and has better tissue penetration into tumor tissues and across the blood-brain barrier. SdAb, with its excellent characteristics, has been widely used in the fields of scientific research, diagnosis, and therapeutic treatment.

In September 2018, the world's first sdAb drug was approved for marketing by the FDA, which is a milestone event that further boosted sdAb R&D and industrialization fever.

Highlights
* SdAb is easily captured on magnetic beads by His tagging, and the antigen-binding domain is exposed to the liquid surface, which contributes to antigen capture.
* SdAb has a higher binding surface capacity and a lower non-specific binding rate. It can be eluted under mild conditions, and its high stability allows for its reuse.
* SdAb serves as a crystallization chaperone to lock proteins in a specific conformation, which is used in protein crystallization studies.
* SdAb is expressed intracellularly and forms binders directly to antigens for the development of new research tools in molecular and developmental biology.
* SdAb is expressed intracellularly. For example, sdAb-based systemic immunolabeling visualizes systemic neuronal projections in adult mice to assess the effects of trauma on the central nervous system.

Uses
SdAb captures trace antigens with high density and firmly binding to solid-phase carriers, reduces non-specific binding of Fc, increases sensitivity and specificity of immunological assays, and is preferred for the development of in vitro diagnostic methods. For example, sdAb targeting EGFR has been used in the diagnosis of breast, ovarian, and prostate cancer. ELISA-based methods can detect body fluid antigens. Combined with molecular imaging technology, it can be used for target discovery, effectiveness evaluation, etc. SdAb can also be used as a non-invasive molecular imaging tracer to study the disease process. SdAb, as a probe for novel biosensors with high sensitivity, can be used in the fields of pharmaceuticals, environment, and food analysis.

Of course, the wider application of sdAb is in the fields of disease treatment and drug discovery. SdAb's own high affinity and specificity make it easier to bind to the receptor target on cancer cells with low immunogenicity. Modification of sdAb into drug carriers can achieve active targeting of sdAb-based anticancer drugs to tumor tissues and increase the accumulation of drugs in tumors. SdAb-conjugate drugs utilize sdAb-specific targeting to deliver drugs to the tumor site, interfering with DNA replication in the nucleus and inhibiting the proliferation of cancer cells. The sequence of sdAb is more similar to the human heavy chain variable region, and the humanization process is simpler. CAR-T with humanized sdAb as the targeting structural domain has been used in several clinical studies.

Although sdAb has obvious advantages, it still faces challenges in practical application, especially in preparation. Phage display technology is commonly used to construct sdAb libraries to screen and enrich specific sdAb, and the preparation process is relatively complex and requires more optimization. There are various biotech companies specializing in sdAb development currently.

Over the past 30 years, traditional monoclonal antibodies have occupied an important position in the development of therapeutic biomolecule drugs. In 1993, Belgian scientists published a paper in Nature, discovering for the first time in the alpaca body a naturally occurring variable region of heavy-chain antibody (HcAb) lacking light chain with a molecular weight of only 15 kDa, which is 1/10 of the molecular weight of traditional antibodies.  It is also the smallest known natural antibody, and it is named a single domain antibody (sdAb).

Due to its unique advantages of a small structural domain, good stability, flexible route of administration, high solubility, and microbial expression, sdAb has gradually become an emerging force in the new generation of therapeutic biopharmaceuticals and clinical diagnostic reagents. In addition, sdAb is more likely to recognize antigens that cannot be captured by traditional antibodies and has better tissue penetration into tumor tissues and across the blood-brain barrier. SdAb, with its excellent characteristics, has been widely used in the fields of scientific research, diagnosis, and therapeutic treatment.

In September 2018, the world's first sdAb drug was approved for marketing by the FDA, which is a milestone event that further boosted sdAb R&D and industrialization fever.

Highlights
* SdAb is easily captured on magnetic beads by His tagging, and the antigen-binding domain is exposed to the liquid surface, which contributes to antigen capture.
* SdAb has a higher binding surface capacity and a lower non-specific binding rate. It can be eluted under mild conditions, and its high stability allows for its reuse.
* SdAb serves as a crystallization chaperone to lock proteins in a specific conformation, which is used in protein crystallization studies.
* SdAb is expressed intracellularly and forms binders directly to antigens for the development of new research tools in molecular and developmental biology.
* SdAb is expressed intracellularly. For example, sdAb-based systemic immunolabeling visualizes systemic neuronal projections in adult mice to assess the effects of trauma on the central nervous system.

Uses
SdAb captures trace antigens with high density and firmly binding to solid-phase carriers, reduces non-specific binding of Fc, increases sensitivity and specificity of immunological assays, and is preferred for the development of in vitro diagnostic methods. For example, sdAb targeting EGFR has been used in the diagnosis of breast, ovarian, and prostate cancer. ELISA-based methods can detect body fluid antigens. Combined with molecular imaging technology, it can be used for target discovery, effectiveness evaluation, etc. SdAb can also be used as a non-invasive molecular imaging tracer to study the disease process. SdAb, as a probe for novel biosensors with high sensitivity, can be used in the fields of pharmaceuticals, environment, and food analysis.

Of course, the wider application of sdAb is in the fields of disease treatment and drug discovery. SdAb's own high affinity and specificity make it easier to bind to the receptor target on cancer cells with low immunogenicity. Modification of sdAb into drug carriers can achieve active targeting of sdAb-based anticancer drugs to tumor tissues and increase the accumulation of drugs in tumors. SdAb-conjugate drugs utilize sdAb-specific targeting to deliver drugs to the tumor site, interfering with DNA replication in the nucleus and inhibiting the proliferation of cancer cells. The sequence of sdAb is more similar to the human heavy chain variable region, and the humanization process is simpler. CAR-T with humanized sdAb as the targeting structural domain has been used in several clinical studies.

Although sdAb has obvious advantages, it still faces challenges in practical application, especially in preparation. Phage display technology is commonly used to construct sdAb libraries to screen and enrich specific sdAb, and the preparation process is relatively complex and requires more optimization. There are various biotech companies specializing in sdAb development currently.

Primary glioblastoma (GBM) is a highly aggressive central nervous system (CNS) cancer that is challenging to treat, with a median survival of 12–15 months despite multimodal treatment regimens. GBM is highly diffuse and infiltrates the normal brain parenchyma, which makes it impossible to surgically remove the tumor, and residual tumors will inevitably recur. In addition, GBM has significant tumor heterogeneity, with subpopulations of cells displaying different mutations, copy number aberrations, gene expression patterns, and epigenetic status. This heterogeneity renders treatment targeting specific molecular processes ineffective, as it can generate tumor recurrence from clones with different genetic compositions.

Temozolomide (TMZ) is currently the first-line chemotherapeutic agent for GBM, and the methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter determines the sensitivity to TMZ treatment. Although TMZ-based therapies have shown relatively few side effects and prolonged survival in patients with GBM, the majority of patients will still manage disease progression. At the time of tumor recurrence, 23% of MGMT-silenced GBM patients exhibited hypermutated tumors associated with poor survival. For patients with recurrent astrocytomas and oligodendrogliomas, this percentage is even higher. TMZ increases the incidence of somatic mutations, which, coupled with the instability of the tumor genome and loss of the DNA mismatch repair (MMR) pathway, leads to hypermutation. And critically, there are no effective treatments for hypermutated gliomas. Therefore, there is an urgent need to develop new therapeutic strategies to effectively eliminate GBM cells, regardless of their mutational and epigenetic characteristics.

Recently, a collaboration of researchers from the Gladstone Institutes, the University of California, Berkeley, and other institutions published a paper in Cell Reports entitled: Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis. The study develops a CRISPR-based cancer-crushing method that targets the non-coding repeats specific to cancer cells to remove them for the treatment of glioblastoma.

The CRISPR system, derived from the adaptive immune systems of bacteria and archaea, is capable of introducing RNA-guided DNA double-strand breaks (DSBs) at targeted sites in the genome, allowing programmable gene editing in human cells. And a potential risk of CRISPR therapies is precisely because of the cytotoxic effects that may result from the DNA double-strand breaks they produce.

Previously, George Church, Luhan Yang, and others used CRISPR-Cas9 gene editing to achieve 62 and 25 gene edits in porcine cell lines and primary cells in vivo in living pigs, respectively, in order to construct gene-edited pigs that could be used for human organ transplantation. But we don't yet know how many such DNA double-strand breaks can eliminate a cell and whether this could be utilized as an effective way to fight cancer.

In this latest study, targeting CNS cancers, the team proposes to harness the cytotoxic effects of DNA double-strand breaks generated during CRISPR-Cas9 gene editing by targeting unique repetitive sequences in the tumor genome as a new strategy for treating recurrent hypermutated gliomas.

Specifically, the team identified germ cell defects in the DNA damage response (DDR) gene that may contribute to the high mutational load of primary GBM tumors, as well as somatic mutations in the TERT promoter, which is critical for the immortalization of GBM cells. Furthermore, recurrent GBM is genetically distinct from primary GBM, with the former having additional mutations associated with malignant progression and hypermutation.

The study also found that recurrent GBM hypermutations induced by treatment with TMZ, a first-line chemotherapeutic agent for GBM, can result in unique, cancer-specific, non-coding repeat sequences. Importantly, these non-coding repeats can be targeted by CRISPR-Cas9, which cleaves the cancer cell genome at these sites, fragmenting the genome and leading to DNA damage and cell death, thereby selectively removing patient-derived recurrent GBM cell lines while preserving normal cells.

This CRISPR-based cancer smashing approach presents an innovative therapeutic paradigm that is independent of the genetic and epigenetic origins of tumors, translating tumor mutational load and TMZ signaling from chemotherapeutic agents in hypermutated cancers into a potential therapeutic pathway.

Primary glioblastoma (GBM) is a highly aggressive central nervous system (CNS) cancer that is challenging to treat, with a median survival of 12–15 months despite multimodal treatment regimens. GBM is highly diffuse and infiltrates the normal brain parenchyma, which makes it impossible to surgically remove the tumor, and residual tumors will inevitably recur. In addition, GBM has significant tumor heterogeneity, with subpopulations of cells displaying different mutations, copy number aberrations, gene expression patterns, and epigenetic status. This heterogeneity renders treatment targeting specific molecular processes ineffective, as it can generate tumor recurrence from clones with different genetic compositions.

Temozolomide (TMZ) is currently the first-line chemotherapeutic agent for GBM, and the methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter determines the sensitivity to TMZ treatment. Although TMZ-based therapies have shown relatively few side effects and prolonged survival in patients with GBM, the majority of patients will still manage disease progression. At the time of tumor recurrence, 23% of MGMT-silenced GBM patients exhibited hypermutated tumors associated with poor survival. For patients with recurrent astrocytomas and oligodendrogliomas, this percentage is even higher. TMZ increases the incidence of somatic mutations, which, coupled with the instability of the tumor genome and loss of the DNA mismatch repair (MMR) pathway, leads to hypermutation. And critically, there are no effective treatments for hypermutated gliomas. Therefore, there is an urgent need to develop new therapeutic strategies to effectively eliminate GBM cells, regardless of their mutational and epigenetic characteristics.

Recently, a collaboration of researchers from the Gladstone Institutes, the University of California, Berkeley, and other institutions published a paper in Cell Reports entitled: Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis. The study develops a CRISPR-based cancer-crushing method that targets the non-coding repeats specific to cancer cells to remove them for the treatment of glioblastoma.

The CRISPR system, derived from the adaptive immune systems of bacteria and archaea, is capable of introducing RNA-guided DNA double-strand breaks (DSBs) at targeted sites in the genome, allowing programmable gene editing in human cells. And a potential risk of CRISPR therapies is precisely because of the cytotoxic effects that may result from the DNA double-strand breaks they produce.

Previously, George Church, Luhan Yang, and others used CRISPR-Cas9 gene editing to achieve 62 and 25 gene edits in porcine cell lines and primary cells in vivo in living pigs, respectively, in order to construct gene-edited pigs that could be used for human organ transplantation. But we don't yet know how many such DNA double-strand breaks can eliminate a cell and whether this could be utilized as an effective way to fight cancer.

In this latest study, targeting CNS cancers, the team proposes to harness the cytotoxic effects of DNA double-strand breaks generated during CRISPR-Cas9 gene editing by targeting unique repetitive sequences in the tumor genome as a new strategy for treating recurrent hypermutated gliomas.

Specifically, the team identified germ cell defects in the DNA damage response (DDR) gene that may contribute to the high mutational load of primary GBM tumors, as well as somatic mutations in the TERT promoter, which is critical for the immortalization of GBM cells. Furthermore, recurrent GBM is genetically distinct from primary GBM, with the former having additional mutations associated with malignant progression and hypermutation.

The study also found that recurrent GBM hypermutations induced by treatment with TMZ, a first-line chemotherapeutic agent for GBM, can result in unique, cancer-specific, non-coding repeat sequences. Importantly, these non-coding repeats can be targeted by CRISPR-Cas9, which cleaves the cancer cell genome at these sites, fragmenting the genome and leading to DNA damage and cell death, thereby selectively removing patient-derived recurrent GBM cell lines while preserving normal cells.

This CRISPR-based cancer smashing approach presents an innovative therapeutic paradigm that is independent of the genetic and epigenetic origins of tumors, translating tumor mutational load and TMZ signaling from chemotherapeutic agents in hypermutated cancers into a potential therapeutic pathway.

Monkeypox, a zoonotic disease, was first discovered in monkeys used for research in 1958 and is spread primarily among animals. The first human case of monkeypox was found in the Democratic Republic of Congo (DRC) in 1970, and since then, monkeypox has been endemic to a number of African countries, including Nigeria, the DRC, and the Central African Republic. Monkeypox cases outside of Africa are usually contracted as a result of traveling to Africa.

In the first half of 2022, monkeypox began to spread in Europe and North America, and on July 23, 2022, WHO Director General Tandace declared the monkeypox outbreak to be a "Public Health Emergency of International Concern" (PHEIC). The 2022 outbreak has caused 8,000 cases in more than 110 countries and territories around the world. The 2022 outbreak has caused more than 80,000 infections in more than 110 countries and territories worldwide, including approximately 30,000 cases in the United States.

Unlike previous monkeypox outbreaks, this one has spread rapidly around the world and is predominantly prevalent among men who have sex with men. The strain responsible for this monkeypox outbreak has been identified as the West African B.1 spectrum branch IIb monkeypox virus.

On September 20, 2023, researchers at Harvard Medical School published a research paper in the journal Cell entitled "Mpox infection protects against re-challenge in rhesus macaques".

The study demonstrated that natural immunity induced by all three routes of infection with the monkeypox virus—intravenous, intradermal, and rectal injections—induced protective efficacy against re-challenge of the monkeypox virus. These observations provide mechanistic insights into the pathogenesis and immunity of monkeypox. In addition, this study demonstrates the usefulness of this non-human primate model for testing monkeypox vaccines and treatments.

In the context of the current monkeypox epidemic, our understanding of monkeypox virus pathogenesis and immunity remains limited. In particular, the current monkeypox outbreak is spreading primarily among men who have sex with men (MSM) populations, and it is not clear whether this nontraditional route of infection leads to natural immunity to prevent reinfection after infection and then tract. And this information is critical to the development of vaccine strategies, epidemiological modeling, and public health approaches related to the vaccine.

To explore this question, the research team constructed a model of monkeypox virus-infected rhesus monkeys using the current outbreak strain of monkeypox and evaluated the virologic, immunologic, histopathologic, transcriptomic, and proteomic features of acute infection and protective immunity to reinfection.

The team infected 18 rhesus monkeys by intravenous, intradermal, and rectal routes of injection and observed robust antibody responses and T-cell responses in rhesus monkeys following all three routes of infection. Substantial skin lesions and high plasma monkeypox virus loads were observed after infection by the intravenous and intradermal routes. Skin lesions peaked on day 10 post-infection and subsided on day 28 post-infection.

On day 28, the team infected all recovering rhesus monkeys and three previously uninfected rhesus monkeys with the monkeypox virus. The results showed that all recovering rhesus monkeys were protected from re-infection with monkeypox. Transcriptomics studies showed that upon initial infection with monkeypox virus, the activation of innate immune responses and inflammatory responses as well as T- and B-cell signaling were markedly upregulated, collagen formation and extracellular matrix organization were downregulated, and T-cell and plasma cell responses were rapidly activated, which provided new insights into the pathogenesis of acute monkeypox.

In response to monkeypox virus reattack, innate and inflammatory signals were markedly reduced, whereas T-cell and plasma cell signals were activated very rapidly, suggesting that immune memory cells and humoral immune responses may be critical to the protective effect against reattack.

Overall, these observations provide mechanistic insights into the pathogenesis and immunity of monkeypox. In addition, the study demonstrates that this non-human primate model is useful for testing monkeypox vaccines and treatments.

These data provide key mechanistic insights into monkeypox pathogenesis and immunity and also demonstrate that this non-human primate model is useful for evaluating monkeypox vaccines and therapeutics.