FG88 - Novel monoclonal antibody with therapeutic utility

Description:

FG88 is a family of monoclonal antibodies (mAbs); FG88.2 and FG88.7 are both murine IgG3ƙ mAbs and CH88.2 and CH88.7 are IgG1 chimerics, all having been generated from NUTACs novel glycolipid platform. The mAbs bind specifically to their target on both glycolipids and glycoproteins. They have a relatively restricted normal tissue distribution but retain their strong reactivity with a wide range of solid tumours. The mAbs have the ability to induce both direct cell killing, without the need for immune effector cells, as well as synergising with antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytolysis (CDC), and have completely cured established, metastatic tumours in pre-clinical models. The mAbs ability to eradicate well established tumours indicates their potential as monotherapeutic agents for the treatment of multiple human solid tumours. Cellular internalisation of the mAbs has also been observed using saporin-conjugated anti-mouse/human IgG (ZAP) and via confocal microscopy, suggesting they have a role to play in antibody drug conjugates (ADCs).

Our USP

Glycolipids are co-accessory molecules involved in regulation of many physiological processes and inhibition of these leads to rapid cell death. They represent ideal targets for novel cancer therapeutics as they are over-expressed by tumours compared to normal tissues and, as glycolipids are densely clustered, mAbs binding them are excellent mediators of immune effector functions. The Nottingham University Therapeutic Antibody Centre (NUTAC) has recently developed a novel platform for producing mAbs targeting glycolipids. These mAbs demonstrate a high degree of binding specificity for their target, allowing us to redefine the normal expression of these glycans and thus reduce toxicity but retain efficacy.

Profile

The FG88 mAbs were raised against tumour glycolipids. These mAbs were subsequently shown to bind glycans on both glycolipids and glycoproteins. The FG88 mAbs bind with very high density to 74% of pancreatic tumours, 69% of colorectal, 56% of gastric, 23% of lung, 27% of breast and 31% of ovarian tumours but have restricted normal tissue binding. The mAbs induced direct cell death by a process related to oncosis/necrosis and completely cure established metastatic, tumours in pre-clinical models.

Defining the epitopes being recognised using the Consortium for Functional Glycomics array

The recognition of carbohydrate antigens as good therapeutic targets has led to the production of a number of anti- glycan mAbs being raised. However, despite being presented as specific many are frequently observed to cross react with other B blood group, H-type 2 or Lewis antigens. Conversely, the FG88 mAbs recognise a unique cell surface glycan. When the mAbs were sent to the Consortium for Functional Glycomics and screened against ≥600 natural and synthetic glycans to determine their degree of specificity, although having screened over 100 mAbs, none have shown such restricted profiles as those produced by NUTAC.

Tumour tissue binding studies

A variety of tumour types were evaluated for the frequency and intensity of binding. FG88 demonstrated strong reactivity with a wide range of tumour cell lines, and solid tumours on tissue microarrays (TMAs) including colorectal (69%), breast (27%), ovarian (31%), gastric (56%), lung (23%) and pancreatic (74%). Interestingly, whilst only 27% of the 902 breast tumour tissues stained, 34% were triple negative breast cancer (TNBC) and 32% of tumours had a basal phenotype.

Figure 1: Immunohistochemistry tumour tissue binding by FG88. Examples of a) negative, b) weak, c) moderate and d) strong staining on tumour TMAs are shown.

Normal tissue binding studies

The normal tissue binding pattern for FG88.2 and FG88.7 is similar, although interesting not identical. FG88.2 only binds subpopulations of cells within the oesophagus, gall bladder, ileum, jejunum, liver, thymus, small intestine, colon, tonsil and pancreas; whereas, FG88.7 also binds rectum (Figure 2).

Figure 2: Representative photomicrographs of normal tissue specimens stained with FG88.

The mouse and chimeric mAbs bind at subnanomolar concentrations to Lewis A and Lewis A expressing cells (Figure 3A & B). Binding of the mAbs inhibits cellular proliferation and induced pore formation in cells (Figure 3C).

 

(A)

 

(B)

(C)

Figure 3: Binding of FG88.2 and CH88.2 at subnanomolar concentrations to Lewis A (A) and the Lewis A expressing colorectal cell line, C170 (B). Scanning electron microscope (SEM) analysis of C170 cells incubated with the mAbs demonstrates pore formation in cells (C); a-b) medium alone, c-d) 0.4% saporin, e-f) FG88.7 (30μg/ml), g-h) FG88.2 (30μg/ml) and i-j) 0.5% hydrogen peroxide for 20hrs at 37 degrees C. Magnifications are at x2000 (bar=10μm) and x10,000 (bar=1μm).

Pre-clinical anti-tumour responses

The mAbs have the ability to induce ADCC and CDC. In addition, they have also demonstrated direct killing of tumour cell lines via a non-apoptotic mechanism of action which is reminiscent of oncosis. The ability of an antibody to induce direct cell death of its target has the potential to synergise with ADCC and CDC mechanisms to give rise to an increased and potent therapeutic effect. FG88 has demonstrated anti-tumour activity in an established therapeutic metastatic colorectal tumour model (Figure 4). Tumour was implanted i.p. and allowed to metastasise to the liver before treatment with mAb (100μg twice weekly). The mAbs cured bulky disease. The potential of the mAbs in eradicating well established tumours indicates their potential as monotherapeutic agents for the treatment of multiple target expressing human solid tumours.

Figure 4: Anti-tumour activity of FG88 in an established therapeutic metastatic colorectal tumour pre-clinical tumour model. Analysis by Log Rank Mantel-Cox test demonstrates significant survival by FG88 (p=0.0037) when compared to the vehicle control. Dosing of the control mAb group was halted early.

Antibody internalisation

Antibody drug conjugates (ADCs) combine the specificity of a mAb with the potency of cytotoxic molecules; among all the antibody-related products, ADCs have become increasing important for oncology therapeutics. Cellular internalisation of the FG88 mAbs has been observed using confocal microscopy (Figure 5).

Figure 5: Internalisation and subcellular localisation of FG88 and CH88 mAbs into C170 cells as observed by confocal microscopy. FG88 and CH88 mAbs were labelled by Alexa FluorÒ 488 dye (495/519nm). C170 cells were incubated with 5μg/ml of labelled FG88 and CH88 mAbs for 1hr and then labelled with Hoechst nucleic acid stain (1μg/ml; 350/461nm), LysoTrackerÒ Deep Red Lysosomal stain (50nM; 647/668nm) and CellMaskOrange Plasma Membrane Stains (2.5μg/ml; 554/567nm). Merged images taken every 10min showing labelled FG88 and CH88 mAbs internalised into C170 cells and co-localised with lysosomes (white arrows). Magnification x60.

Internalisation and cytotoxicity of drug conjugates has been demonstrated with saporin-conjugated anti-mouse/human IgG (ZAP; Figure 6) suggesting they may have a role to play with ADC technology.

      

Figure 6: FG88.2, FG88.7 and CH88.2 internalisation data over a range of concentrations (0.01nM to 10nM) on the colorectal cell line, C170. Cellular internalisation of the mAbs was observed using 75ng/well saporin-15 conjugated goat antibody (F’ab fragment) to mouse (ATSbio, IT#48) or human IgG (ZAP) and assessed by inhibition of thymidine uptake (added for the last 24hrs of incubation). Control wells consisted of cells incubated with culture medium only, with the irrelevant positive control IgG3 mAb FG88 and with secondary conjugate only.

Intellectual Property

A patent application for the target and the FG88 mAbs has been submitted.

Nottingham University Therapeutic Antibody Centre (NUTAC)

NUTAC is a new initiative pump primed by the University of Nottingham to develop therapeutic mAbs to cancer, specifically to tumour associated glycans. The Centre is based on Professor Lindy Durrant’s track record of delivering innovative immunotherapeutic products to the clinic. The lead anti-glycolipid mAb, CEP-37250/KHK2804, is currently in Phase I/II clinical trials in the US for patients with colorectal and pancreatic cancer in a joint development between Teva and Kyowa Hakko Kirin Pharma Inc.

Contact:

Professor Lindy Durrant – Head of NUTAC
Tel: +44 (0)115 8231863; email: lindy.durrant@nottingham.ac.uk

Dr Tina Parsons – Business Manager
Tel: +44 (0)115 8231863; email: tina.parsons@nottingham.ac.uk

http://www.nottingham.ac.uk/nutac

Patent Information:
Category(s):
Healthcare
Cancer
For Information, Contact:
Caroline Sykes
IP Commercialisation Manager - Healthcare
The University of Nottingham
0115 82 30042
Caroline.Sykes@nottingham.ac.uk
Inventors:
Lindy Durrant
Jiaxin Chua
Tina Parsons
Keywords:
© 2017. All Rights Reserved. Powered by Inteum