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Bispecific Monoclonal Antibody

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HuiYing Teh

on 20 May 2014

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Transcript of Bispecific Monoclonal Antibody

Bispecific Monoclonal Antibody
Research Team
BISPECIFIC MONOCLONAL ANTIBODY
Three types of bispecific antibodies: trifunctional antibody, chemically linked Fab and bi-specific T-cell engager .
Parts of the two different antibodies are coloured blue and green, respectively.
INTRODUCTION
T cells are among the most potent cytotoxic cells in humans.

The receptor on T cells that confers the specificity of their response to foreign antigen in association with a self histocompatibility antigen has recently been identified.

Two chains, each of 40-50 kDa, called a and 13, form a disulfidebonded heterodimer.

Both chains possess variable (V) and constant (C) regions that are encoded by separate V region, diversity (D) region, joining (J) region, and C region genes

This heterodimer is noncovalently associated on the cell surface with three invariant polypeptides referred to as the T3 complex

More than two decades of work on bispecific antibodies to direct T cells to attack cancer cells has occurred
The development of an effective and scalable drug format has proved difficult and undergone multiple iterations.
Target antigens explored for tumor therapy with bispecific antibodies:
Differentiation antigens such as CD19, CD33, CEA, EpCAM, HER-2/neu, PSMA, or EGF receptor.
Overexpressed on cancer cells relative to normal cells such as HER 2/neu or EGFR
Safe depletion of the entire cell compartment along with cancer cells such as CD19 or CD33
Accessible on cancer cells but are sequestered on normal tissues (such as CEA or EpCAM).

PROBLEM STATEMENT
Using recombinant antibody-based technologies to engage a polyclonal population of cytotoxic T cells.

To find a powerful therapeutic approach to treat or prevent cancer

Although mAbs have been found to target specific antigens, numerous impediments are emerged in cancer therapy by mAbs because of expression of a low level of the same antigens on the surface of normal cells.

A novel strategy to target cancer cells more selectively which has an affinity against the tumor-specific antigens.

The mutations in gene profile could create novel tumor-specific proteins and receptors which could be detected.

OBJECTIVES
To target cancer cells specifically and decrease the side effects of antibodies in targeted cancer immunotherapy

Novel idea against the tumor-specific antigens caused by mutations in chromosomes.

Intends to consider a novel multivalent bsmAbs to target epitopes on the same antigen

T CELL TARGETING
What?
A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen.
REFERENCES
Frankel, S. R., & Baeuerle, P. A. (2013). Targeting T cells to tumor cells using bispecific antibodies.
Current Opinion in Chemical Biology,
17(3), 385-392.
Staerz, U. D., & Bevan, M. J. (1986). Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity.
Proceedings of the National Academy of Sciences of the United States of America
, 83(5), 1453-1457.
Target cell
S.AKR , T-cell lymphoma derived from an AKR/J mouse that expresses H-2k and the Thy-1.1 alloantigens.
EL4 , T-cell lymphoma derived from a C57BL/6 mouse that expresses H-2b and Thy-1.2.
-Both lymphomas are maintained in culture in RPMI 1640 medium containing 10o fetal bovine serum.

Mab
F23.1 hybridoma , fusion of immunized C57L/J spleen cells with the P3 myeloma (15).
- The C57L/J spleen donor had been immunized four times with nylon wool-purified T cells of BALB.B mice.

- Isolate a line of F23.1 that was HAT-sensitive and ouabain-resistant that continued to produce the mAb. This line was fused using polyethylene glycol to the 19E12 hybridoma (HAT-resistant, ouabain-sensitive) in a 1:1 ratio.

PURIFICATION &
CHARACTERIZATION

Separation of the "parental" immunoglobulins, F23.1 and 19E12, from the hybrid antibody was achieved by chromatography on a hydroxylapatite HPLC column(Bio-Gel HPHT, Bio-Rad)

Chemical characterization of the hybrid and parental immunoglobulins was performed by isoelectric focusing using Pharmalyte 5-8 (Pharmacia).

PREPARATION OF RECOMBINANT F(AB’)

F(ab')2 fragments of both antibodies were reduced with 10 mM mercaptoethanol at pH 6.8 and the column-purified Fab' monomers were recombined at pH 8 under a stream of oxygen

CTL
ASSAY

100ul of medium containing 1 x 104 Na251CrO4-labeled target cells (S.AKR or EL4) and 3 x 104 CTL clone (OE4 or OE25) was added to each well.
Plates were incubated at 37°C for 4 hr and centrifuged, and 100 /4 of the supernatant was removed and assayed for radioactivity.
Spontaneous release was measured by culturing labeled target cells in the absence of CTL.

METHOD
EXPECTED RESULT
RESEARCH BUDGET
Teh Hui Ying (167131)
Chai Siew Ying (167527)
Ain Zakiah bt Zaharullail (169250)
Pang Chin Lin (169158)
Amir Hamzah b. Ahmad (160141)

Result Summary
Total Expenses RM 60,000

Return of Investment 100%

Payback year 3 years
Limitation of Monoclonal Antibody
Lack of natural Fc regions which may delay antobody clearance an improves pharmacokinetic properties.
Cannot activate T-lymphocytes
Many patients do not adequately respond to monospecific therapy

Advantage of Bispecific Monoclonal Antobody
Ability to combine a cytotoxic cell (eg: CD3) or ADC with a tumour specific protein target (eg: CD 19 or CD 20)
ability of engaging T-cells for tumor cell elimination
Easier for Human body to pick up the antibody
Bispecific antibody fragments are expressed as a single polypeptide chain they include potentially immunogenic linkers
Full transcript