Antibody-Derivative Biotherapeutics: Fragments and Fusions Define the Future

Cheryl Scott

February 9, 2022

19 Min Read


ON THE COVER: Artist’s rendering of heavy-chain–only antibodies with variable domains in light purple and conventional immunoglobulins with variable domains in light green. (HTTPS://STOCK.ADOBE.COM)

Monoclonal antibodies (MAbs) remain the dominant biopharmaceutical product class, but as biotechnologies have advanced in recent decades, developers have found ways to exploit their “magic-bullet” capabilities while putting aside their limitations. That has led to a new generation of antibody-related therapeutics created by cutting and pasting molecular domains. Researchers are mixing and matching functional moieties of antibodies and other molecules to create custom-designed proteins with powerful efficacy and tunable targeting. A simple search at Taylor & Francis Online (, the academic journal portal of BPI’s parent company, turns up hundreds of citations involving antibody fragments, conjugates, and bi-/multispecific molecules from the past year alone. Many articles, some of which are described below, are open-access and otherwise freely available for further research.

Antibody-Engineering Platforms
A number of biopharmaceutical innovators and researchers are working on platform technologies for designing therapeutic bispecific/multispecific antibodies. Below are three examples published in mAbs during 2021: one from industry and two from academia.

Targeting Cancer: Authors from the Roche Innovation Centers in Germany and Switzerland reviewed the progress of their company’s CrossMab technology for bispecific antibody (bsAb) development (1). About 10 years ago, Roche introduced this technology for building correct antibody light-chain and heavy-chain associations. The technology has evolved and expanded in application since then, bringing several bispecifics from Roche and others into development and clinical trials. After reintroducing the technology, the authors highlight its applications for generating both bi- and multispecific antibodies with different geometries and mechanisms of action, then review the CrossMab-based therapeutics that are currently in development (1).

Researchers at the University of Toronto in Canada and the University of Chicago in Illinois have worked together to engineer a new bsAb format — dual-antigen T-cell engagers (DATEs) — by fusing single-chain variable fragments (scFvs) that target the B-cell membrane protein CD3 to a tumor-targeting antigen-binding fragment (2). The purpose is to direct T cells to specific features of solid-cancer cells. The authors report success in using a number of novel paratopes against different tumor antigens to recruit T-cell cytotoxicity to tumor cells both in vitro and in an in vivo xenograft model of pancreatic ductal adenocarcinoma. Because unique surface antigens in solid tumors are limited, the team sought to enhance selectivity with “double-DATEs” that target two tumor antigens simultaneously. Each double-DATE contains an additional, autonomous, variable heavy-chain domain that binds a second tumor antigen without eliciting a cytotoxic response itself.

“This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens,” the authors state (2). “The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.”

Scientists at the University of Stuttgart in Germany use a diabody platform for multivalence and multispecifity in oncology applications (3). The laboratory developed a novel bivalent and bispecific antagonistic molecule (Dab-Fc) targeting human epidermal growth factors 2 and 3 (HER2, HER3). A Dab-Fc combines the variable domains of an anti-HER2 antibody with an anti-HER3 antibody in a noncovalent dimer of scFv fragment structure that is stabilized by CH1 and CL constant domains — all fused to a human γ1 Fc region. The resulting “Dab-Fc 2 × 3” molecule exhibits unhindered binding to both HER antigens and binds them sequentially.

In cell-based experiments, the resulting molecule strongly bound to different tumor cell lines and was efficiently internalized, inhibiting tumor-cell proliferation and migration (3). It exhibited IgG-like pharmacokinetics and antitumoral activity in a xenograft tumor model of gastric cancer. “These results illustrate the suitability of our versatile Db-Ig platform technology for the generation of bivalent bispecific molecules,” the authors write, “which has been successfully used here for the dual targeting of HER2 and HER3.”

IgG Subclasses: The four subclasses of human IgG antibodies differ in their constant regions, particularly in their hinges and CH2 domains. IgG1 has the highest binding affinity to Fc receptors for immune regulation, followed by IgG3, IgG2, and IgG4. Most MAb drugs are IgG1 molecules, but scientists from Dartmouth College (Hanover, NH) and Duke University Medical Center (Durham, NC) have made the case for the IgG3 subclass, which is “conspicuously absent” among MAb therapies and Fc-fusion protein biologics.

Despite its high affinity for activating Fcγ receptors, effective complement fixation, and a long hinge that “appears better suited for low-abundance targets,” developers have been put off by IgG3’s potential for rapid degradation, reduced plasma half-life, and increased immunogenicity. But studies of natural immunity and recombinant therapies suggest that many historical roadblocks are no longer relevant. “Collectively, this body of evidence motivates thoughtful reconsideration of the clinical advancement of this distinctive antibody subclass for treatment of human diseases” (4).

Characterization and Developability
Analytical aspects of antibody-derivative development are going to require new technologies and approaches. Several potential options came to light in 2021.

As authors from the University of Michigan (Ann Arbor, MI) write (5), “The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery.” Developers need to identify the most drug-like candidates for in-depth analysis of safety and efficacy. But time and resource requirements limit the number of molecules that can be tested for developability. The authors cite binding specificity as a key biophysical property, with successful antibody drugs showing low levels of nonspecific binding (polyspecificity). Most assays for detecting that property are limited by poor sensitivity, or they require proprietary surface-display methods. Some such assays use complex and poorly defined polyspecificity reagents.

The authors developed a sensitive flow-cytometry assay for evaluating nonspecific antibody interactions without those limitations (5). The PolySpecificity Particle (PSP) assay can be used to evaluate IgGs, multispecific antibodies, and Fc-fusion proteins. It uses micron-sized magnetic beads coated with protein A to capture antibodies that are present in dilute concentrations (<0.02 mg/mL). Flow-cytometry analysis of polyspecificity reagent binding to those beads then enables sensitive detection of differences in nonspecific interactions.

“Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures,” the authors write. They found that using ovalbumin as a reagent provided the best results for an assay that is much more sensitive than standard enzyme-linked immunosorbent assays (ELISAs). “We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.”

Researchers at AstraZeneca (Gaithersburg, MD) are focused on bsAbs for immunotherapy (6). “T-cell–mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune-checkpoint molecules,” they write. Because bispecifics have complex structures and mechanisms of action (MoAs) that often involve more than one signaling pathway, new bioassays are needed for measuring their potency and other properties. The authors developed a dual-target, cell-based reporter assay for a bsAb that binds human cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed cell-death protein 1 (PD-1) and targets two signaling pathways to negate T-cell activation. The bioassay measures the potency of both antigen target arms simultaneously.

“This dual-target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute (CQA) assessment, and biological properties characterization of the CTLA-4/PD-1 bsAb,” the authors write. “Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the bsAb.” So the method should reflect potential MoAs better than single-reporter assays can, making it applicable to evaluation of other bsAbs and antibody combination therapies.

Another group at AstraZeneca reported on fragmentation degradation of therapeutic antibodies, a CQA typically monitored with capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) (7). CH2 domains linked by intrachain disulfide bonds can break to form fragments — a phenomenon that can be detected by reduced reversed-phase liquid chromatography with mass spectrometry (RP-LC-MS) and reduced CE-SDS methods. However, fragment separation in nonreduced CE-SDS (nrCE-SDS) has not been reported previously, although many scientists believed that fragments would comigrate with intact IgG under such conditions.

After observing a shoulder peak in nrCE-SDS from stability samples of an IgG-like bsAb, the authors determined it to indicate specific fragments. Subunit LC-MS analysis verified that the crystallizable fragment contained variants with one or more additions of ~18 Da, which further investigation revealed to be CH2 clippings largely caused by proteolytic activity. “Cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied,” the authors report (7). “Our study shows that CH2 domain cleavages with complementary fragments still linked by intrachain disulfide can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.”

bsAbs require greater engineering and involve more manufacturing complexity than are found in production of conventional MAbs. bsAbs also can present increased immunogenic risk because they differ from endogenous immunoglobulins and contain new epitopes. A team at Genentech, a member of the Roche group (South San Francisco, CA) is working on an “anti-A/B” bispecific designed using the familiar “knobs-into-holes” (KIH) format (8). When the bsAb exhibited unexpectedly high immunogenicity in both preclinical and clinical studies, the company terminated that clinical program. Developers then used an integrated approach combining in silico analysis, in vitro assays, and an in vivo study in nonhuman primates to characterize the product’s immunogenicity. Results indicated that it was associated with epitopes in the anti-B arm, not with mutations engineered through the KIH process. “Our results showed the value of this integrated approach for performing immunogenicity risk assessment during clinical candidate selection to effectively mitigate risks during bsAb development” (8).

Immunotherapy Strategies
Several product approvals have confirmed the value/validity of immunotherapeutic approaches to cancer treatment, and more promising candidates are advancing through the development pipeline. A number of companies are using bsAbs and other fusion proteins for this type of therapeutic intervention — and academic researchers continue to push the concept further by focusing on T-cell engagement and other pathways.

T-Cell Engagers: Researchers from Teneobio in Newark, CA (now part of Amgen) and the University of Southern California (Los Angeles, CA) have examined the therapeutic potential of targeting CD19 in B-cell malignancies (9). The approach already has produced encouraging clinical data with T-cell–targeting agents such as blinatumomab and chimeric antigen receptor (CAR)-T therapies for acute lymphoblastic leukemia and B-cell non-Hodgkin’s lymphoma (B-NHL). However, both approaches also have demonstrated nonideal PKs and significant toxicities, as well as manufacturing challenges.

The Tenebio–USC team developed a fully human CD19–CD3 bsAb (TNB-486) that could address the limitations of approved treatments for B-NHL treatment (9). The authors show that TNB-486 induces tumor cell lysis in vitro, with minimal cytokine release in the presence of both CD19+ target cells and T cells. And it clears CD19+ tumor cells in immunocompromised mice with human peripheral blood mononuclear cells (PBMCs) in multiple models. The candidate’s PK profiles in mice and cynomolgus monkeys resemble those of conventional antibodies. “This new T-cell–engaging bsAb targeting CD19 represents a novel therapeutic that induces potent T-cell–mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.”

Meanwhile, scientists at Roche Innovation Centers in Switzerland and Germany are working with others at Radboud University Medical Center (Nijmegen, the Netherlands), Wake Forest University School of Medicine (Winston-Salem, NC), and the University of Turin (Italy) on an immunocytokine fusion protein that combines an antibody against fibroblast activation protein α (FAP) with an interleukin (IL-2) variant (10). The authors investigated its immunostimulatory properties in combination with PD-1 checkpoint inhibition, CD40 agonism, a T-cell bispecific, and antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC).

Through in vitro and in vivo studies, the interdisciplinary team found that the FAP-IL2v molecule induced dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells but was less potent at activating immunosuppressive regulatory T cells (Tregs). Imaging studies showed good tumor targeting. FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate both antibody-dependent or T-cell–dependent cellular cytotoxicity (TDCC) and programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with anti-PD-L1 and agonistic CD40 antibodies was most efficacious overall. The authors conclude, “These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies” (10).

Alternative Approaches: Two European companies — Affimed (Heidelberg, Germany) and Arjuna Therapeutics (Santiago De Compostela, Spain) — are working together on cancer therapeutics targeting epidermal growth factor receptor (EGFR). In their 2021 report, they point out that both MAbs and tyrosine kinase inhibitors with demonstrated clinical efficacy are limited in two ways (11): toxicity causing a narrow therapeutic window and resistance mechanisms in some cancers through the EGFR signaling cascade. Based on a proprietary redirected, optimized cell-killing (ROCK) antibody platform, the collaborators developed a novel bispecific IgG1-scFv fusion antibody called AFM24 to target both EGFR on tumor cells and CD16A on innate immune cells (rather than on T cells).

In vitro, AFM24 is highly potent and effective for ADCC through the action of NK cells, and it also mediated antibody-dependent cellular phagocytosis by macrophages. The team showed their bsAb to be effective against a number of EGFR-expressing tumor cells regardless of their EGFR expression levels and mutational status.

In vivo, AFM24 was well tolerated by cynomolgus monkeys at doses ≤75 mg/kg (administered once weekly for 28 days), with no skin or organ toxicities seen. The monkeys showed transient elevation of IL-6 levels soon after administration and returned to baseline after 24 hours. “These results emphasize the promise of bispecific innate-cell engagers as an alternative cancer therapy,” the authors write, “and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status” (11).

Scientists at Chulalongkorn University (Bangkok, Thailand) are studying α folate receptor (FRα) as a target for treating patients with non–small-cell lung cancer (NSCLC). FRα is highly expressed in tumor cells but largely absent in normal tissues. In their 2021 report, the authors describe enrichment and selection by phage-display “bioplanning” of a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα (12). When ELISA testing identified a candidate with specific FRa binding, the team moved on to express it in Escherichia coli as a soluble protein. It showed high affinity for FRα, with specific binding to both FRα-expressing NSCLC cells (standards) and NSCLC patient-derived primary cancer cells. The fragment also was internalized into those cells. The authors conclude, “This study inspires the use of phage display to develop human VH Ab fragments that might be well suited for drug-targeted therapy of NSCLC and other FRα-positive cancer cells” (12).

Scientists at Southwest Medical University (Luzhou, China) and its affiliated hospital (Sichuan, China) reported in 2021 on the results of another phage-display screening method (13). They used an immune scFv library from the PBMCs of patients with different types of malignant tumors. Because many epithelial-derived malignancies show enhanced expression of ephrin type-A receptor 2 (EphA2), it is considered to be an important target for antitumor therapy. Therapeutic MAbs against such immune checkpoints have shown good efficacy for tumor treatment.

“High-affinity scFvs against EphA2 can be easily screened from the immune library using phage-display technology,” the authors write. They explain that anti-EphA2 scFvs can be modified into different forms of therapeutic fusion molecules to improve their affinity for EphA2. The team demonstrated binding activity of the resulting recombinant antibodies to the EphA2 protein, tumor cells, and tumor tissues using macromolecular interaction techniques, flow cytometry, and immunohistochemistry. Constructed scFvs-Fc and IgG1 antibodies inhibited tumor-cell growth to some extent.

“These results suggest that the immune libraries from patients with malignant tumors are more likely to screen for antibodies with high affinity and therapeutic effect,” the authors conclude (13). “The constructed fully human scFv immune library has broad application prospects for specific antibody screening. The screened scFv-Fc and IgG1 antibodies against EphA2 can be used for the further study of tumor immunotherapy.”

Pandemic Solutions
SARS-CoV-2 has dominated headlines and life science for nearly two years now. The global health crisis and its economic tolls have necessitated myriad viral mitigation strategies, and vaccines are just one of those. So it’s no surprise to find many published reports of innovative work using antibody fragments and fusions for COVID-19 testing and treatment. Below are just three examples from industry and academia.

As I discovered in preparing a recent BPI eBook, timely point-of-care diagnosis is important to saving lives and resources during a pandemic (14). Authors from Mologic (Thurleigh, UK) and Lancaster University (Lancaster, UK) reviewed the industry’s progress in applying recombinant antibody fragments to diagnostics (15). “There is intense pressure for more accurate and less expensive rapid diagnostic tests with a value preferably <$1,” the authors report. Manufacturers need large-scale, cost-effective production of recombinant antibodies, ideally expressed by microbial hosts.

The review highlights E. coli expression for use in production of rapid, inexpensive tests — detailing genetic engineering strategies and associated challenges. The authors emphasize the importance of expression scale and culture parameters to expression titers. Large-scale production provides for greater cell densities and higher yields: A shake-flask culture can provide 10–20 mg/L of functional antigen-binding antibody fragments (Fabs), bioreactor yields can be 1–2 g/L, and 10–500 mg of such proteins are needed to accommodate a million rapid tests. “Despite the substantial importance of the production of the antibodies and their fragments,” the authors caution, “their downstream processing should be appropriately considered from the beginning for achieving the target value of the final rapid diagnostic tests.”

Scientists at the University of California–San Francisco and the Chan Zuckerberg Biohub (San Francisco, CA) are focused on treatment with neutralizing antibodies that target SARS-CoV-2. Most such products in development directly block the binding of viral spike receptor-binding domains (RBDs) to angiotensin-converting enzyme II (ACE2). This team is using nonneutralizing RBD antibodies that can assist in neutralization when they are linked to neutralizing binders (16).

“We identified Fabs by phage display that bind RBD but do not block ACE2 or neutralize virus as IgGs,” the authors report. “When these nonneutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~25-fold potency improvement in neutralizing SARS-CoV-2 compared to the monospecific bivalent VH-Fc alone or the cocktail of the VH-Fc and IgG.” The effect was epitope-dependent, reflecting a unique structural geometry. Results show that a bsAb that combines both neutralizing and nonneutralizing epitopes on the spike-RBD provides a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies (16).

Meanwhile, Janssen Biotherapeutics (Spring House, PA) and Alector (South San Francisco, CA) researchers have turned their interest to a novel delivery approach (17). Because SARS-CoV-2 infection usually begins in nasal mucosa, and the virus propagates mainly at that site throughout the course of most patients’ disease, this team wants to block the virus there. Mucosal administration of biologics has presented substantial difficulty for many years, however.

The authors describe bifunctional molecules combining single-domain variable regions that bind to both the polymeric immunoglobulin receptor (pIgR) and the viral spike protein through addition of an ACE2 extracellular domain (ECD). They hypothesize that pIgR will transport the resulting bispecific from circulation to the mucosal surface, where the ECD could act as a decoy virus receptor. In studies using human tissue, the bifunctional molecules can move across lung epithelia without inducing ADCC against pIgR-expressing cells. “These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa” (17).

Bispecifics, Fragments, and Antibody Fusions
In this supplement, BPI contributes to the expanding literature of antibody fragments and fusions with a review of bispecifics in development and two technical reports related to fragments and fusion proteins. The former comes from our friends at The Antibody Society. One of the others describes Abbott’s use of disposables in microbial production of antibody fragments for diagnostic applications. The other highlights a potential fusion partner for MAb-based immunotherapies.

1 Surowka M, Schaefer W, Klein C. Ten Years in the Making: Application of CrossMab Technology for the Development of Therapeutic Bispecific Antibodies and Antibody Fusion Proteins. mAbs 13(1) 2021: 1967714:

2 Enderle L, et al. A T Cell Redirection Platform for Co-Targeting Dual Antigens on Solid Tumors. mAbs 13(1) 2021: 1933690;

3 Rau A, et al. A Bivalent, Bispecific Dab-Fc Antibody Molecule for Dual Targeting of HER2 and HER3. mAbs 13(1): 1902034;

4 Chu TH, Patz Jr. EF, Ackerman ME. Coming Together at the Hinges: Therapeutic Prospects of IgG3. mAbs 13(1) 2021: 1882028;

5 Makowski EK, et al. Highly Sensitive Detection of Antibody Nonspecific Interactions Using Flow Cytometry. mAbs 13(1) 2021: 1951426;

6 Chen W, et al. Development of a Mechanism of Action-Reflective, Dual Target Cell-Based Reporter Bioassay for a Bispecific Monoclonal Antibody Targeting Human CTLA-4 and PD-1. mAbs 13(1) 2021: 1914359;

7 Cao M, et al. Identification of a CE-SDS Shoulder Peak As Disulfidelinked Fragments from Common CH2 Cleavages in IgGs and IgG-Like Bispecific Antibodies. mAbs 13(1) 2021: 1981806;

8 Cohen S, et al. An Integrated Approach for Characterizing Immunogenic Responses Toward a Bispecific Antibody. mAbs 13(1) 2021: 1944017;

9 Malik-Chaudhry HK, et al. TNB-486 Induces Potent Tumor Cell Cytotoxicity Coupled with Low Cytokine Release in Preclinical Models of B-NHL. mAbs 13(1) 2021: 1890411;

10 Waldhauer I, et al. Simlukafusp Alfa (FAPIL2v) Immunocytokine Is a Versatile Combination Partner for Cancer Immunotherapy. mAbs 13(1) 2021: 1913791;

11 Wingert S, et al. Preclinical Evaluation of AFM24, a Novel CD16A-Specific Innate Immune Cell Engager Targeting EGFR-Positive Tumors. mAbs 13(1) 2021: 1950264;

12 Parakasikron N, et al. Development of a Human Antibody Fragment Directed Against the Alpha Folate Receptor As a Promising Molecule for Targeted Application. Drug Deliv. 28(1) 2021: 1443–1454;

13 Yang Y, et al. Fully Human Recombinant Antibodies Against EphA2 from a Multi-Tumor Patient Immune Library Suitable for Tumor-Targeted Therapy. Bioengineered 2021;

14 Scott C, Beck V, Bergamin F. Diagnostics: Developing Rapid and Accessible Testing Solutions. BioProcess Int. eBook December 2021:

15 Huleani S, et al. Escherichia coli As an Antibody Expression Host for the Production of Diagnostic Proteins: Significance and Expression. Crit. Rev. Biotechnol. (2021);

16 Lim SA, et al. Bispecific VH/Fab Antibodies Targeting Neutralizing and Non-Neutralizing Spike Epitopes Demonstrate Enhanced Potency Against SARS-CoV-2. mAbs 13(1) 2021: 1893426;

17 White I, et al. Bifunctional Molecules Targeting SARS-CoV-2 Spike and the Polymeric Ig Receptor Display Neutralization Activity and Mucosal Enrichment. mAbs 13(1) 2021: 1987180;

Cheryl Scott is cofounder and senior technical editor of BioProcess International, part of Informa Connect; 1-212-600-3429; [email protected].

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