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Antibody based therapy was firstly developed in the 1970s following the discovery of the structure of antibodies and the development of hybridoma technology,

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Presentation on theme: "Antibody based therapy was firstly developed in the 1970s following the discovery of the structure of antibodies and the development of hybridoma technology,"— Presentation transcript:

1 Antibody based therapy was firstly developed in the 1970s following the discovery of the structure of antibodies and the development of hybridoma technology, which provided the first reliable source of monoclonal antibodies. These advances allowed for the specific targeting of tumors both in vitro and in vivo. Initial research on malignant neoplasms found monoclonal antibody (mAb) therapy efficient with solid tumors, but, of limited and generally short-lived success with blood malignancies. Nevertheless, the field of immunotherapy never stopped for a moment and also currently is very progressive. Four major antibody types were developed: murine, chimeric, humanized and human. Antibodies of each type are distinguished by suffixes on their name. Murine Initial therapeutic antibodies were murine (suffix -omab). Murine antibodies were obtained by hybridoma technology, for which Jerne, Köhler and Milstein received a Nobel prize (1984). However the dissimilarity between murine and human immune systems led to the clinical failure of these antibodies, except in some specific circumstances. These antibodies: 1. Raised an immune response against them resulted with a short half-life in vivo, the formation of immune complexes after repeated administration, mild allergic reactions and sometimes even an anaphylactic shock. 2. limited penetration into tumor sites. 3. Inadequately recruitment of host effector functions.  Hybridoma cells growing in tissue culture. The image shows a single clone of cells each of which is producing large amounts of a specific monoclonal antibody which the cells secrete and which can be readily purified from the culture media.(using protein A/G agarose beads) (1)  Immunization of a mouse. (2  Isolation of B cells from the spleen. (3)  Cultivation of myeloma cells. (4)  Fusion of myeloma and B cells. (5) Separation of cell lines by dilution (1/3 cell/96W well). (6)  Screening of suitable cell lines. (7)  in vitro (a) or in vivo (b) multiplication. (8)  Harvesting.

2 To reduce murine antibody  immunogenicity, murine molecules were engineered to remove immunogenic content and to increase immunologic efficiency. This was initially achieved by the production of chimeric (suffix -ximab) and humanized antibodies (suffix -zumab). Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Taking human gene sequences from the kappa light chain and the IgG1 heavy chain results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increases serum half-life. Humanized antibodies are produced by grafting murine hypervariable regions on amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin. Humanized antibodies usually bind antigen much more weakly than the parental murine monoclonal antibody, with reported decreases in affinity of up to several hundredfold. Increases in antibody-antigen binding strength have been achieved by introducing mutations into the complementary determining regions (CDR) using techniques such as chain-shuffling, randomization of complementarity-determining regions and antibodies with mutations within the variable regions induced by error-prone  PCR, E. coli mutator strains and site specific mutagenesis. Complementarity-determining regions (CDRs) are part of the variable chains in  immunoglobulin antibodies and T-cell receptors, where these molecules bind to their specific antigens. Since most sequence variation associated with immunoglobulins and T-cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. The sketch defines an antibody with the variable domains shown in blue, and the CDRs (which are part of the variable domains) in light blue.

3 Monoclonal Abs production using phage display
Antibody Phage Display: Technique and Applications - Christoph M. Hammers, Christoph M. Hammers Journal of Investigative Dermatology, 2014

4 The key to success is preparation of quality RNA from the cell source chosen (e.g., peripheral blood mononuclear cells). This RNA is reverse-transcribed into cDNA, which is used for PCR of the VH and VL chains of the encoded antibodies (Figure 1a, 1b). Defined sets of primers specific for the different VH and VL chain–region gene families then allow amplification of all transcribed rearranged variable regions within a given immunoglobulin repertoire for library construction, thus reflecting all antibody specificities in a particular individual. This immortalizes recombinant cDNA clones for expressed Igs. Variations on phage display libraries include (i) libraries constructed for Ig isotypes (e.g., IgG, IgA, and IgE) and (ii) libraries of mAbs expressed as Fab fragments or as single-chain variable fragments (scFv), the latter of which consist of the VH and VL joined by a linker 

5 Helper phages (e.g. M13K07) provide all the necessary gene products for particle formation when using phagemid vectors. They are mutated wild‐type phage containing the whole genome, with a defective origin of replication or packaging signal, and hence, are inefficient in self‐packaging. When infecting E. coli containing a phagemid, phage progeny is produced with only the phagemid being packaged efficiently. Therefore, the resulting phage virion contains a mixture of wild‐type and fusion coat protein, and the genetic information of the fusion protein is encoded by the packaged phagemid. The VH and VL PCR products, representing the antibody repertoire, are ligated into a phage display vector (e.g., the phagemid pComb3X) that is engineered to express the VH and VL as an scFv fused to the pIII minor capsid protein of a filamentous bacteriophage ofEscherichia coli that was originally derived from the M13 bacteriophage. However, the phage display vector pComb3X does not have all the other genes necessary to encode a full bacteriophage in E. coli. For those genes, a helper phage is added to the E. coli that are transformed with the phage display vector library. The result is a library of phages, each expressing on its surface a mAb and harboring the vector with the respective nucleotide sequence within. (Figure 1c). In addition to the ability to produce phage displaying the mAb, the phage display vector can be used to produce the mAb itself (not attached to phage capsid proteins) in certain strains of E. coli. Additional cDNA is engineered, in the phage display vector, after the VL and VH sequences to allow characterization and purification of the mAb produced. Specifically, the recombinant antibody may have a hemagglutinin (HA) epitope tag and a polyhistidine to allow easy purification from solution (Barbas, 2001).

6 Assembly of combinatorial antibody libraries on phage surfaces: The gene III site
Carlos F. et al 1991 FIG. 2. Electron micrographs showing antigen-specific labeling of filamentous phage displaying Fab molecules. (A) Filamentous phage as produced with the pComb 3 phagemid system display tetanus tox~oid-specific Fab molecules on their tail. Phage were labeled with 5- to 7-nm colloidal gold particles coated with tetanus toxoid (x140,OO0.) (B) Filamentous phage, as previously reported using the gVIII multivalent display system (11), shows antigenspecific labeling along the body of the phage and is provided for comparison. (x65,000.)

7 Diverse APD libraries are produced from ~108 independent E
Diverse APD libraries are produced from ~108  independent E. coli transformants infected with helper phage. A library is screened for phage binding to an antigen through its expressed surface mAb by a technique called (bio-) panning. Cyclic panning allows for pulling out potentially very rare antigen-binding clones and consists of multiple rounds of phage binding to antigen (immobilized on ELISA plates or in solution on cell surfaces), washing, elution, and reamplification of the phage binders in E. coli. During each round, specific binders are selected out from the pool by washing away nonbinders and selectively eluting binding phage clones. After three or four rounds, highly specific binding of phage clones through their surface mAb is characteristic for directed selection on immobilized antigen. For panning on eukaryotic cell surfaces, more rounds of panning are usually needed, and more sophisticated protocols involving cell-sorting techniques have been published. Of note, it is also possible to perform double recognition panning to select for bispecific mAbs (i.e., mAbs that recognize two antigens), as demonstrated in a patient with active mucocutaneous pemphigus vulgaris (PV) and serum antibody reactivity against desmoglein (Dsg) 3 and Dsg1, yielding scFv specific for both Dsg3 and Dsg1.

8 After cyclic panning, the resultant polyclonal (i. e
After cyclic panning, the resultant polyclonal (i.e., a mixture of all the phages that bind to the antigen chosen) phage pools are used to infect E. coli. Infected bacteria is plated out and individual colonies are picked and expanded for monoclonal phage production. These are each tested again by phage ELISA to confirm antigen binding. The phage display vector, isolated from each clone, is then subjected to sequencing to determine the nucleotide sequence of VL and VH encoding the mAb that bound to the antigen. Furthermore, soluble scFv (or Fab) from clones of interest can easily be produced in bacteria that have been transformed with the phage display vector of interest. These mAb are then purified by metal chelation (e.g., through polyhistidine) or affinity purification (e.g., through a HA tag). To further analyze these soluble mAbs, a vast array of methods exists .Obtained nucleotide sequences can be analyzed and grouped (e.g., by heavy- or light-chain gene usage and shared “fingerprints,” known as complementarity-determining region 3, indicating common B-cell clonal origin) with tools available online.

9 Microscopic image of direct immunoflurorescence using an anti-IgG antibody. The tissue is skin from a patient with Pemphigus vulgaris. Note the intercellular IgG deposits in the  epidermis and the early intraepidermal  vesicle caused by acantholysis. Pemphigus is a rare group of blistering autoimmune disease that affect the skin and mucos membranes. In pemphigus, autoantibodies form against Desmoglein. Desmoglein forms the "glue" that attaches adjacent epidermal cells via attachment points called Desmosomes. When autoantibodies attack desmogleins, the cells become separated from each other and the epidermis becomes "unglued", a phenomenon called  acantholysis. This causes blisters that slough off and turn into sores. In some cases, these blisters can cover a significant area of the skin. Originally, the cause of this disease was unknown, and "pemphigus" was used to refer to any blistering disease of the skin and mucosa. In 1964, researchers found that the blood of patients with pemphigus contained antibodies to the layers of skin that separate to form the blisters. In 1971, an article investigating the autoimmune nature of this disease was published. Monovalent mAbs (in the form of scFv) cause blister formation typical of pemphigus foliaceus in human skin. After injection of purified antidesmoglein 1 scFv, human skin specimens were cultured for 24 hours. Histology and direct immunofluorescence (IF) are shown. 3-07/1e and 3-30/3h caused superficial epidermal blisters. All scFv, except 3-094/O1808, bound to the cell surface of epidermal keratinocytes. 

10 Px44-hTRAIL binds to human epidermal keratinocytes and causes apoptosis of tumor cells. A) When injected intradermally into human skin, the fusion protein binds to the cell surface of epidermal keratinocytes as detected by immunofluorescence with anti-hemagglutinin (HA) and anti-hTRAIL antibodies. B) TUNEL staining of actinic keratosis-like lesions in K14-activated Fyn transgenic mice after intratumoral injection of the fusion protein shows apoptosis of tumor cells (green fluorescence signal). hTRAIL, human tumor necrosis factor–related apoptosis-inducing ligand. Reprinted with permission from Kouno et al, 2013. In a translational research approach designed to use a nonpathogenic cloned scFv from a pemphigus patient to deliver a biologically active agent to the epidermis, Kouno et al (2013) created a fusion protein containing Px44, a nonpathogenic anti-Dsg-scFv domain linnked to an active domain of human tumor necrosis factor–related apoptosis-inducing ligand (hTRAIL). The recombinantly produced Px44-hTRAIL fusion protein bound and delivered TRAIL to the cell surface of human keratinocytes (5a) The TRAIL component maintained its known biological activity both to inhibit secretion of γ-interferon by activated CD4+ T cells and to cause apoptosis of rapidly proliferating lymphocytes and keratinocytes. In a preliminary study Px44–hTRAIL caused apoptosis in a mouse model of actinic keratosis and early squamous-cell carcinoma (5b). These studies suggest that mAbs cloned from pemphigus patients may be useful as agents to deliver biologically active proteins to the epidermis for therapy of various immunological and neoplastic diseases.

11 Inhibitory receptors 11

12 12

13 Anti human-CD300a/anti-human-IgE
Developing an Anti human-CD300a/anti-human-IgE Bispecific antibody! 13

14 Full antibody in vitro system
Anti-IgE Anti-CD300a Cross linker 14

15 (Whole ab. In vitro system)
The “Modular” System (Whole ab. In vitro system) H-IgE M-a-H-IgE Human Mast cell FceRI G-a-M f(ab)2 M-a-H-CD300a CD300a

16 Inhibition of Mast cell activation by M-a-H-CD300a
Clone E59 (Morreta’s) commercial. d5/8 Tryptase activity (O.D. 410nm units)

17 Inhibition of Mast cell activation by M-a-H-CD300a
Hybridoma (Offer’s) sups. d24/8B Tryptase activity (O.D. 405nm units)

18 Chemical synthesis of aCD300a/aH-IgE Bispecific ab.
8/03/2010 pepsinized IgG I αCO300a Column: Superose 12 prep. 97 x 1.6cm (~195ml) - 4ml/fract.

19 8/03/2010 peptinized II α IgE reduction Column: Superose 12 prep
8/03/2010 peptinized II α IgE reduction Column: Superose 12 prep. 97 x 1.6cm (~195ml) - 4ml/fract.

20 11/03/2010 peptinized II α IgE reduction Column: Superose 12 prep
11/03/2010 peptinized II α IgE reduction Column: Superose 12 prep. 97 x 1.6cm (~195ml) - 4ml/fract.

21 PAGE analysis of Bispecific ab. synthesis step by step
Silver staining Pooled and concentrated Frac.-TNB pooled and concentrated frac.-SH Pooled and concentrated Frac. aCD300a parental aCD300a digested aIgE parental aIgE digested frac. 16 frac. 19 frac. 16 frac. 19 frac. 15 frac. 16 frac. 17 frac. 18 frac. 19 frac. 20 frac. 21 M M 250 250 150 150 100 100 75 75 50 50 37 37 25 25

22 PAGE analysis of Bispecific ab. Hybrid and Mix Coomassie blue staining
F(ab)2 hybrid F(ab)’ mix M Intact ab. 250 Hybrid aCD300a/a-H-IgE f(ab)2 150 100 75 50 Mix aCD300a f(ab)’ + aH-IgE f(ab)’ 37 25

23 The “Direct” System – using Bispecific ab.
M-a-H-IgE Or: Rabb.-a-H-IgE (activating) H-IgE Human Mast cell FceRI Bispecific ab. aH-IgE/aCD300a (inhibition) CD300a

24 Tryptase activity (O.D. 405nm units)
Direct inhibition of Mast cell activation by aCD300a/aH-IgE Bispecific ab. 100% 68% 67% 75% d19/1 Tryptase activity (O.D. 405nm units)


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