Bispecific antibody applications can be broadly split into practical, diagnostic and therapeutic.
Practical Application of Bispecific Antibodies
One of the earliest uses of bispecific antibodies was in cell purification protocols developed by StemCell Technologies. Here, whole antibodies are linked together using a linker linking the constant regions of the two antibodies. This is a totally different type of bispecific antibody compared to those that were later developed for diagnostic and therapeutic purposes.
Here’s how a StemCell cell purification protocol typically uses bi-specific antibodies.
- Let’s say we’d like to purify untouched CD4+ T cells from human blood so we target the remaining cells using StemCell’s bispecific monoclonal antibody (mAbs) cocktail customized for human CD4+ T cell enrichment.
- One mAb in the bispecific antibody targets antigens expressed by non-T blood cells.
- Each bi-specific antibody pair also contains a mAb against Dextran, a polysaccharide.
- Non-T cells become coated with these bi-specific mAbs.
- Next, Dextran-coated iron nanoparticles bind the anti-Dextran mAb in the bi-specific antibodies.
- When sample tube is placed in a magnet, Dextran-coated iron nanoparticles get stuck to the tube wall in response to the magnetic field.
- So mAb-bound cells also bound to the Dextran-coated iron nanoparticles get stuck to the tube wall as well.
- Unbound cells are poured off. Most of them are untouched CD4+ T cells.
Using different custom bispecific antibody cocktails, a variety of cell types can be enriched from a variety of sources including blood, lymphoid tissue, tissue culture, etc.
Engineered bispecific antibodies used in diagnostics and cancer therapeutics
For diagnostics and cancer therapeutics, engineering approaches using bacterial (E. coli) or mammalian recombinant expression systems are used to synthesize bispecific antibodies from scratch.
How are engineered Bispecific Antibodies used in Diagnostics?
Advantages of engineered Bispecific Antibodies used in Diagnostics
- In traditional ELISA (Enzyme-linked Immunosorbent Assay),
- mAbs are linked to detecting enzymes like HRP randomly.
- Batch-to-batch variation.
- Site of linkage could block antibody’s binding site, promote its aggregation, reduce its antigen affinity.
- BsmAb-detection enzyme linkage, OTOH, is always 1:1 because linked to a half-, not full, antibody
- Unlike traditional ELISA mAb, linkage done during ab purification step, negating later enzyme addition and washing off.
- Much more specific than traditional ELISA.
- Reduced false-positive results and background noise.
- Improved signal-to-noise ratio, simpler and faster.
Disadvantages of engineered Bispecific Antibodies used in Diagnostics
- Few diagnostic BsmAbs currently available.
- Multiple BsmAb generation approaches means varying degrees of difficulty in terms of yield and need for purification.
- Uniformity of approach needed.
- Recombinant formats would help increase number of diagnostic BsmAbs.
Bispecific antibody Diagnostic Applications
Bacteria: Bordetellapertussis (1), Escherichiacoli O157:H7 (2), Mycobacterium tuberculosis (3), Staphylococcus aureus (preliminary; 4).
Viruses:Dengue (5, 6), Hepatitis B: Specific but less sensitive than traditional ELISA, more useful as primary Point-of-Care (POC) test (7), SARS (8).
Cancer:For circulating prostate-specific antigen (9), In vivo cancer diagnostic imaging (in mouse models) (10).
Engineered Bispecific Antibodies used in Cancer Therapeutics
Dozens of BsmAbs generated using at least 6 different engineering approaches are currently in human Phase I or II studies.
Two BsmAbs have been approved so far for human use, namely, Blinatumumab and Catumaxomab.
Blinatumumab (Blincyto®, AMG 103, MT 103,MEDI 538)
- Developed and licensed by Amgen.
- Approved in the USA in 2014 for Precursor B cell ALL (Acute Lymphoblastic Leukemia).
- Stands for B lineage-specific anti-tumor mouse monoclonal antibody.
- As is typical for most modern drugs approved for human use, it took more than 15 years to get to market.
- Uses the BiTE (Bispecific T cell Engager) approach. What is BiTE?
- Essentially, one half-antibody piece binds a T cell-specific surface molecule, CD3. The other half-antibody piece binds a tumor-specific antigen, in this case, a B cell tumor-specific surface molecule, CD19.
- This brings T cells in close proximity to the targeted tumor cell, forcing an immunological synapse between T cell and tumor.
- T cell gets activated. If it’s a tumor-specific CD8 cytotoxic T cell, it will release cytotoxic granules into the tumor cell, killing it.
Advantages of the BiTE approach
Advantages of Blinatumumab
- Unlike natural antibodies, Blinatumumab is a single-chain protein where the antigen-binding portions of the two different antibodies are linked by a non-immunogenic linker that endows it with the flexibility to rotate such that it can bind to different antigenic epitopes on two separate cell types in close proximity (11).
- Its single-chain structure gives it several advantages: easy to synthesize in large amounts, purification is relatively simple.
- In 20 adults with CD19+ ALL, who relapsed or had persistent disease post-chemotherapy, 16/20 (80% response) and 61% relapse-free survival at median 33 months follow-up (12, 13).
- 25/36 adults (69%) with relapsed/refractory ALL had either complete or partial hematological remission (14).
- CD19 is expressed by most B cell lineage malignancies but absent on hematopoietic stem cells and plasma cells so relatively good tumor target specificity.
- Signaling pathway associated with CD19 molecule, the PI3 kinase pathway, is implicated in survival and resistance to chemotherapy in hematologic malignancies so such tumors may likely retain CD19 expression even under the selective pressure of therapy (15).
Disadvantages of Blinatumumab
- Recruited T cells are not tumor-specific. The anti-CD3 mAb part of the BsmAb binds all types of T cells.
- Has short half-life (16).
- In ALL trials, it was given continuously intravenously for 28 days (13, 14).
- Patients need to be pre-medicated with dexamethasone (a corticosteroid, i.e. immune suppressant) within 1 hour of the 1st drug infusion (17).
- Side-effects include flu-like symptoms (chills, fatigue, fever, headache) that coincided with maximal T cell cytokine release (18)
- Side-effects may be more severe with greater tumor burden (19).
- Other adverse events included neurologic symptoms such as disorientation and seizure. They were fully reversible but led to drug stoppage in some patients (20).
- Dose-dependent effect.
- Better in patients with minimal residual disease compared to relapsed/refractory B cell precursor ALL.
- Licensed by Fresenius Biotech (Trion Pharma).
- Approved in the EU in 2009 for Rx of malignant ascites in EpCAM (Epithelial cell Adhesion Molecule)-positive tumors.
- Generated using one of the oldest methodologies. Basically, two hybridomas, each specifically secreting a mAb, were fused to make a hybrid hybridoma or quadroma.
- The quadroma secretes a mixture of mAbs and the BsmAb.
- The BsmAb is purified from this mixture.
- One mAb is mouse IgG2a, specific for human CD3, a T cell-specific surface molecule.
- Other mAb is rat IgG2b, specific for EpCAM.
- The Fc portion of this BsmAb can bind to the activatory Fc receptors, Fc gamma I, IIa and II.
- EpCAM, a type I transmembrane protein, is expressed by many epithelial cells.
- Tumor EpCAM expression different from normal. Latter is baso-lateral and masked by tight junctions. In tumors, OTOH, it’s expressed all over the cell surface, making it more easily available for binding.
- EpCAM is frequently expressed by many epithelial cell tumors, especially those that cause malignant ascites such as colon, endometrial, gastric, lung, ovarian, pancreatic and prostate.
- Malignant ascites is peritoneal fluid accumulation.
- This happens when malignant cells spread to the peritoneal cavity.
- Thus, approved for late-stage tumor associated with poor prognosis.
How does Catumaxomab work?
- Currently we understand some processes, not others.
- For example, most patients develop anti-mouse, and -rat antibodies after Catumaxomab Rx.
- Yet, these ADA (anti-drug antibodies) correlate with a more, not less, favorable outcome (21). Why? Not known.
- In the key PhaseII/III study (n=258), necessity for fluid removal was increased from 11 days to 46 days after Catumaxomab Rx (22).
- Overall survival in gastric cancer patients was 77 versus 44 days.
- Responses included fever, and transient GI tract and liver function disturbances.
- Tang, X. L., et al. “Use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for Bordetella pertussis.” Clinical and diagnostic laboratory immunology 11.4 (2004): 752-757.
- Guttikonda, Sujatha, et al. “Monospecific and bispecific antibodies against E. coli O157 for diagnostics.” Journal of immunological methods 327.1 (2007): 1-9.
- Sarkar, Susmita, et al. “A bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings.” PloS one 7.2 (2012): e32340.
- Wagstaffe, Sarah J., et al. “Bispecific antibody-mediated detection of the Staphylococcus aureus thermonuclease.” Analytical chemistry 84.14 (2012): 5876-5884.
- Ganguly, Advaita, et al. “Heterosandwich immunoswab assay for dengue virus Ns1 antigen detection.” Diagnostic microbiology and infectious disease 78.1 (2014): 35-39.
- Ganguly, Advaita, et al. “PRODUCTION AND CHARACTERIZATION OF MONOSPECIFIC AND BISPECIFIC ANTIBODIES AGAINST DENGUE VIRUS NS1 PROTEIN.” Journal of virological methods (2015).
- Chen, Yu-Ping, et al. “Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen.” Clinical and Vaccine Immunology 14.6 (2007): 720-725.
- Severe Acute Respiratory Syndrome) (Kammila, Sriram, et al. “A rapid point of care immunoswab assay for SARS-CoV detection.” Journal of virological methods 152.1 (2008): 77-84.
- Kreutz, Fernando T., and Mavanur R. Suresh. “Novel bispecific immunoprobe for rapid and sensitive detection of prostate-specific antigen.” Clinical chemistry 43.4 (1997): 649-656.
- Byrne, Hannah, et al. “A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications.” Trends in biotechnology 31.11 (2013): 621-632.
- Löffler, Anja, et al. “A recombinant bispecific single-chain antibody, CD19× CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes.” Blood 95.6 (2000): 2098-2103.
- Topp, Max S., et al. “Targeted therapy with the T-cell–engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival.” Journal of clinical oncology 29.18 (2011): 2493-2498.
- Topp, Max S., et al. “Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL.” Blood 120.26 (2012): 5185-5187.
- Topp, Max S., et al. “Phase II Trial of the Anti-CD19 Bispecific T Cell–Engager Blinatumomab Shows Hematologic and Molecular Remissions in Patients With Relapsed or Refractory B-Precursor Acute Lymphoblastic Leukemia.” Journal of Clinical Oncology (2014): JCO-2014.
- Khwaja, Asim. “PI3K as a target for therapy in haematological malignancies.” Phosphoinositide 3-kinase in Health and Disease. Springer Berlin Heidelberg, 2011. 169-188.
- Wolf, Evelyn, et al. “BiTEs: bispecific antibody constructs with unique anti-tumor activity.” Drug discovery today 10.18 (2005): 1237-1244.
- Oak, Eunhye, and Nancy L. Bartlett. “Blinatumomab for the treatment of B-cell lymphoma.” Expert opinion on investigational drugs 0 (2015): 1-10.
- Klinger, Matthias, et al. “Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell–engaging CD19/CD3-bispecific BiTE antibody blinatumomab.” Blood 119.26 (2012): 6226-6233.
- Baeuerle, Patrick A., and Carsten Reinhardt. “Bispecific T-cell engaging antibodies for cancer therapy.” Cancer research 69.12 (2009): 4941-4944.
- Zimmerman, Zachary, Tap Maniar, and Dirk Nagorsen. “Unleashing the clinical power of T cells: CD19/CD3 bi-specific T cell engager (BiTE®) antibody construct blinatumomab as a potential therapy.” International immunology (2014): dxu089.
- Ott, Marion G., et al. “Humoral response to catumaxomab correlates with clinical outcome: results of the pivotal phase II/III study in patients with malignant ascites.” International journal of cancer 130.9 (2012): 2195-2203.
- Heiss, Markus M., et al. “The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: results of a prospective randomized phase II/III trial.” International Journal of Cancer 127.9 (2010): 2209-2221.